Sterol Regulatory Elements

ABSTRACT

Disclosed are discrete functionally translocatable DNA segments, termed Sterol Regulatory Elements (SREs), which are fused to heterologous structural genes to provide sterol regulatory capability to the thus formed hybrid gene. The hybrid genes respond to sterols by decreasing the production of messenger RNA. The SRE segments contain as their primary functional nucleotide sequence, a 16 bp sequence referred to as direct repeat 2, isolated from the 5&#39; regions of the human LDL receptor gene. DNA segments which include this 16 nucleotide long sequence similarly confer sterol regulatory capability to previously known promoters such as the HSV TK promoter. Also disclosed are discrete sequences which confer positive transcription promotion to heterologous structural genes and promoters without conferring sterol responsivity. Methods are disclosed for employing these genetic control elements in a myriad of embodiments which provide for a fine-tune control of heterologous genes. Methods are also disclosed for employing the SRE in a screening assay for drugs capable of stimulating the cell to synthesize LDL receptors.

The government may own certain rights in the present invention pursuantto NIH grantL HL 20948.

This application is a continuing application of U.S. Ser. Nos.07/033,302 , now U.S. Pat. No. 4,935,363, and 07/033,081, both filedMar. 30, 1987.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to DNA segments which may be employed asfunctionally translocatable genetic control elements. More particularly,the invention relates to sterol regulatory elements and promotersequences which serve to promote transcription and/or confer asterol-mediated suppression capability to selected structural genes.

2. Description of the Related Art

In the 25 years since Jacob and Monod first proposed the lac operonmodel and the concept of messenger RNA (see, Jacob et al. (1961), J.Mol. Biol., 3: 318-350), the structure and function of a number ofprokaryotic operons has been elucidated in elegant detail. For example,in the case of the lac operon, it has been shown that transcriptionalcontrol of the various structural genes of the operon (e.g.,B-galactosidase) resides in an upstream (i.e., 5' with respect to thestructural genes) regulator gene and operator gene. The regulator geneproduces a protein "repressor" that interacts with the operator toprevent transcription initiation of the structural gene. Inducers suchas IPTG (isopropyl thiogalactoside) bind to the repressor and therebyinduce transcription by preventing the binding of the repressor to theoperator. Additionally, there is a promoter site P, upstream of theoperator and downstream of the regulatory gene, which serves as an RNApolymerase binding site.

Studies on the lac operon further have led to the discovery andelucidation of the mechanism of prokaryotic catabolic suppression. In E.coli it is found that the catabolic suppress presence of glucose in thegrowth medium serves to shut down the expression of gluconeogenicpathways, including the lac operon and its associated structural genes.The mechanism of this catabolic suppression is not entirely clear, butappears to involve a glucose-mediated suppression of cyclic AMP-mediatedstimulation of transcription. In this regard, it appears as thoughcyclic AMP complexes with a protein known as catabolic gene activatorprotein (CAP), and this complex stimulates transcription initiation.Thus, in the presence of glucose, the activator CAP complex is notformed and transcription is not enhanced.

In addition to the lac operon, the mechanism and structure of numerousadditional prokaryotic control mechanisms have been elucidated. (e.g.,see Miller et al. (eds.), 1978, The Operon. Cold Spring HarborLaboratory; Wilcox et al. (1974), J. Biol. Chem., 249: 2946-2952(arabinose operon); Oxender et al. 1979), Proc. Natl. Acad. Sci.,U.S.A., 76: 5524-5528 (trp operon); Ptashne et al. (1976), Science, 194:156-161 (lambda phage)).

Unfortunately, in contrast to prokaryotic systems, very little ispresently known about the control mechanisms in eukaryotic systems.Moreover, although, as noted, the mechanisms for feedback suppression ofmRNA production in prokaryotes have been elucidated in elegant detail(see e.g., Ptashne, M. (1986) A Genetic Switch: Gene Control and PhageLambda. Cell Press and Blackwell Publications, Cambridge, Mass. and PaloAlto, Calif. pp. 1-128), little is known about analogous mechanisms inhigher eukaryotes. In animal cells most attention has focused onpositively-regulated systems in which hormones, metabolic inducers, anddevelopmental factors increase transcription of genes. These inducingagents are generally thought to activate or form complexes with proteinsthat stimulate transcription by binding to short sequences of 10 to 20basepairs (bp) in the 5'-flanking region of the target gene. Theseelements have been called GRE, MRE, or IRE for glucocorticoid regulatoryelement, metal regulatory element, and interferon regulatory element,respectively (Yamamoto (1985), Ann. Rev. Genet., 19: 209-252; Stuart etal. (1984), Proc. Natl. Acad. Sci. USA, 8: 7318-7322; Goodbourn et al. (1986), Cell), 45: 601-610).

Accordingly, there is currently very little knowledge concerningeukaryotic genetic control mechanisms and, in particular, littleknowledge concerning genetic elements controlled by feedback regulation.The availability of discreet DNA segments which are capable ofconferring either a negative or positive control capability to knowngenes in eukaryotic systems would constitute an extremely usefuladvance. Not only would such elements be useful in terms of furtheringour understanding of eukaryotic gene control in general, but would alsoprovide biomedical science with powerful tools which may be employed byman to provide "fine-tune" control of specific gene expression. Theelucidation of such elements would thus provide science with anadditional tool for unraveling the mysteries of eukaryotic gene controland lead to numerous useful applications in the pharmaceutical andbiotechnical industries.

Although the potential applications for such control sequences arevirtually limitless, one particularly useful application would be as thecentral component for screening assays to identify new classes ofpharmacologically active substances which may be employed to manipulatethe transcription of structural genes normally under the control of suchcontrol sequences. For example, in the case of hypercholesterolemia, itwould be desirable to identify therapeutic agents having the ability tostimulate the cellular production of Low Density Lipoprotein (LDL)receptors, which would in turn serve to lower plasma LDL (andconsequently cholesterol) by increasing the cellular uptake of LDL.

Currently, there are few cholesterol-lowering drugs that are both safeand efficacious, and no drugs which are known to operate at theabove-described genetic control level. For example, aside from agentsthat function by sequestering bile salts in the gut and thereby increasecholesterol excretion, the principal therapeutic agent available forcholesterol lowering is Lovastatin, a drug manufactured by Menck, Co.that acts indirectly to stimulate production of LDL receptors.

Specifically, Lovastatin and other drugs in this class (Simvastatin,Pravastatin) act by inhibiting the activity of HMG CoA reductase, therate-limiting enzyme of endogenous cholesterol synthesis. These drugscontain side chains that resemble the native substrate for HMG CoAreductase and thus competitively inhibit the activity of the enzyme.Eventually this lowers the endogenous synthesis of cholesterol and, bynormal homeostatic mechanisms, plasma cholesterol is taken up byincreased LDL receptor populations in order to restore the intracellularcholesterol balance. Conceptually, HMG CoA reductase inhibitors areacting at the penultimate stage of cellular mechanisms for cholesterolmetabolism. It would be most desirable if the synthesis of LDL receptorcould be directly upregulated at the gene level. The upregulation of LDLreceptor synthesis at the gene level offers the promise of resetting thelevel of blood cholesterol at a lower and clinically more desirablelevel

(Brown et al. (1984), Scientific American, 251:58-60). However, nomethods exist for conveniently assaying the ability of a candidatecomposition to exert such an effect on the transcription of the LDLreceptor gene.

Accordingly it is a further object herein to provide a method forconveniently evaluating candidate substances for receptor upregulatingactivity.

SUMMARY OF THE INVENTION

In its most general and overall scope, the present invention is directedto DNA segments which, when located upstream from and proximal to atranscription initiation site of a selected structural gene, serve toconfer a sterol-mediated suppression capability to such a gene. TheseDNA segments, termed Sterol Regulatory Elements (SREs), have beenidentified and constructed from a consideration and manipulation of DNAsequences found in the gene regions upstream of the transcriptioninitiation site of structural genes that are subject to sterol mediatedregulation, such as the HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A)reductase or HMG-CoA synthase genes, or the gene encoding the LDLreceptor protein (Low Density Lipoprotein Receptor). For the purposes ofthe present disclosure, these genes will be referred to collectively asthe "sterol regulated structural genes". Moreover, as used herein, theterm "upstream" refers to DNA sequences found in a 5' direction from agiven point of reference along a DNA molecule.

The LDL receptor gene is the structural gene which provides for theproduction of the LDL receptor protein, the receptor protein responsiblefor the facilitated uptake of cholesterol by mammalian cells. In thecontext of the LDL receptor gene, these SRE sequences provide asterol-mediated regulation of LDL receptor gene transcription. Thus, inthe relative absence of sterols within the cell, transcription of theLDL receptor gene is promoted, whereas in the presence of cholesterol,transcription is suppressed. In contrast to the LDL receptor gene, theHMG-CoA synthase and reductase genes encode enzymes in the cholesterolbiosynthetic pathway. When sterols accumulate within cells, the amountsof mRNA produced by both genes are reduced. Conversely, when sterols aredepleted, the amounts of mRNA rise, and cholesterol synthesis increases.

Most importantly and surprisingly, it has been found that the discreetSRE elements of the present invention are functionally translocatable toother structural genes. Thus, when the SRE are located upstream of aselected heterologous structural gene, sterol-mediated regulation isconferred to this "hybrid" gene. Therefore, as used herein, the term"functionally translocatable" refers to genetic elements which retaintheir functional capability in contexts other than their natural state,and the term "hybrid" gene refers to a man-made gene constructed throughthe application of recombinant DNA techniques to bring together geneticelements not normally associated in nature. Moreover, the term"heterologous structural gene" refers to structural genes other than theLDL receptor gene, and the term "structural gene" refers to any DNAsegment which may be both transcribed and translated by a cell.

Accordingly, in a general sense, the invention is directed to asubstantially purified segment of DNA comprising a functionallytranslocatable sterol regulatory element capable of conferringsterol-mediated regulation of structural gene transcription to aselected heterologous structural gene when located upstream from andproximal to a transcription initiation site of such a gene, providedthat said segment is free of the structural gene ordinarily under thetranscriptional control of said sterol regulatory element, the elementincluding a nucleotide sequence selected from one of the following:

    5'-C-A-C-M-S-Y-A-C-3'

wherein:

Y=C or T

S=C or G

M=C or A;

or the complement of such a sequence.

In more particular embodiments, this DNA segment will be further definedas including a nucleotide sequence selected from one of the following:

    5'-A-T-C-A-C-M-S-Y-A-C-3'

wherein:

Y=C or T

S=C or G

M=C or A;

or the complement of such a sequence.

In still further embodiments, the above described general DNA segmentwill be defined as including within its sequence a nucleotide sequenceselected from one of the following:

    5'-M-M-M-A-T-C-A-C-M-S-Y-A-C-3'

wherein:

Y=C or T

S=C or G

M=C or A;

or the complement of such a sequence.

Moreover, in still further embodiments, the DNA segment of the inventionmay be defined as including within its sequence a nucleotide sequenceselected from one of the following:

    5'-M-M-M-A-T-C-A-C-M-S-Y-A-C-K-K-M-3'

wherein:

K=T or G

Y=C or T

S=C or G

M=C or A;

or the complement of such a sequence.

Generally, a sequence stretch of on the order of 8 to 50 nucleotides inlength, which incorporates one or more of the foregoing sequences, willbe preferred for combination with selected structural genes in order toconfer sterol-mediated regulation. In fact the inventors have determinedthat even shorter segments, on the order of 8 to 27 nucleotides inlength, may be used for conferring such a sterol-mediated control.

In certain particular embodiments, the invention involves a widevariation of particular DNA sequences which may be incorporated intoregions upstream of selected structural genes to bring such structuralgenes under sterol-mediated control. These sequences may be defined asincluding within their sequence, a nucleotide sequence selected from oneof the following:

(a) 5'-A-G-T-T-T-G-C-A-G-T-G-G-G-G-T-G-A-T-T-T-T-C-A -A-A-T-G-3+;

(b) 5'-A-G-T-T-T-G-C-A-G-T-G-G-G-G-T-G-A-T-T-T-T-C-A -A-A-T-G-3';

(c) 5'-A-G-T-C-T-G-C-A-G-T-G-G-T-G-T-G-A-T-T-T-T-C-A -A-A-A-G-3';

(d) 5'-A-G-T-C-T-G-C-A-G-T-G-G-G-G-T-G-A-T-T-T-T-C-A -A-A-A-A-3';

(e) 5'-G-A-G-A-G-A-T-G-G-T-G-C-G-G-T-G-C-C-T-G-T-T-C -T-T-G-G-3';

(f) 5'-G-A-G-A-G-A-T-G-G-T-G-C-G-G-T-G-C-C-T-G-T-T-C -T-T-G-G-3';

(g) 5'-G-A-G-A-G-A-T-G-G-T-G-C-G-G-T-G-C-C-C-G-T-T-C -T-C-C-G-3';

(h) 5'-G-A-G-A-G-A-T-G-G-T-G-C-G-G-T-G-C-C-C-G-T-T-C -T-C-C-G-3';

(i) 5'-A-C-G-G-T-G-A-C-G-T-A-G-G-G-T-G-G-C-C-C-G-A-G -A-A-C-A-3'; or

(j) 5'-A-G-T-G-G-G-G-T-G-A-G-A-C-G-G-T -G-A-C-G-3'; or the complement ofany of these sequences.

In further aspects, the SRE of the present invention refer to discreetDNA segments represented by the formula:

    (X).sub.n

wherein n=1-5, with each X being independently selected from DNAsegments having a nucleotide sequence of:

(a) 5'-A-A-A-A-T-C-A-C-C-C-C-A-C-T-G-C-3'; or

(b) 5'-G-C-A-G-T-G-G-G-G-T-G-A-T-T-T-T-3';

with each X unit, if more than one, being from 0-20 nucleotides selectedfrom the group of nucleotides consisting of A, G, C and T.

It has been found by the inventors that the SRE sequences of the presentinvention confer a sterol-regulatory capability regardless of theirorientation with respect to the transcribed strand of DNA. Moreover, ithas been found that there is no requirement that this sequence be placedin a particular position with respect to the site of transcriptioninitiation. They thus, act in a fashion similar to enhancer elements inthis regard.

It has also surprisingly been determined that the SRE may be introducedinto a heterologous gene in multiple copies, either in a forward orreversed orientation, and thereby obtain a much improved sterolregulatory capability. Moreover, multiple SRE units need not be placedin an adjacent conformation and may be separated by numerous randomnucleotides and still retain their improved regulatory and promotioncapability.

The present invention is also directed to regulatory elements whichserve to confer a promotion of transcription initiation withoutconferring a sterol regulatory capability. As with the SREs, these"positive" promoter sequences are functionally translocatable and may beemployed by locating such sequences upstream from and proximal to atranscription site. In a preferred aspect, the transcription promotersequences are represented by the formula:

    (X).sub.n

wherein n=1-5, each X being independently selected from DNA segmentshaving a nucleotide sequence of:

(a) 5'-A-A-A-C-T-C-C-T-C-C-C-C-C-T-G-C-3';

(b) 5═-G-C-A-G-G-G-G-G-A-G-G-A-G-T-T-T-3';

(c) 5'-A-A-A-C-T-C-C-T-C-C-T-C-T-T-G-C-3'; or

(d) 5'-G-C-A-A-G-A-G-G-A-G-G-A-G-T-T-T-3';

with each X unit, if more than one, being separated by from 0 to 20nucleotides selected from the group of nucleotides consisting of A, G, Cand T.

As noted, the promoter and/or regulatory elements are advantageouslyemployed by locating said sequences upstream from and proximal to atranscription initiation site of a selected heterologous structuralgene. Depending on the particular structural gene employed, thesecontrol elements may provide some benefit when located up to 300nucleotides upstream of a transcription initiation site, as measuredfrom the 3, end of the control sequence.

However, in a preferred embodiment, the sequences are located within b150 nucleotides of transcription initiation.

In a more preferred embodiment, the control sequences are located within100 nucleotides of an initiation site.

In still more preferred embodiments, the control sequences are locatedwithin 50 nucleotides of an initiation site.

Thus, to date, it has been observed that, in general, the closer thecontrol element is to a site of trans-cription initiation, the moreeffective the resultant control.

It is contemplated that the control sequences will prove useful in thecontext of a wide array of genes which have been characterized to date.Although, as disclosed in more detail below, it is believed that thesequences will prove useful in the context of virtually any structuralgene, it is further believed that these sequences will be of particularbenefit in the context of human and related structural genes such as thegenes for t-PA (tissue plasminogen activator), human growth hormone,activin, interferon, lymphokines such as interleukins I and II, tumornecrosis factor, and numerous other genes as disclosed herein.

It is an additional object of the present invention to provide controlsequences which may be combined with known promoters to provide novelhybrid eukaryotic promoters having sterol regulatory capabilities. Suchhybrid promoters may also be employed in the context of selectedheterologous structural genes.

It is, therefore, a particular object of the invention to provide a DNAmolecule which has incorporated within its structure one or more of theDNA sequences or segments having a sterol regulatory element such asdescribed above, in combination with a structural gene not ordinarilyunder the transcriptional control of said element, said structural geneand sterol regulatory element being combined in a manner such that saidstructural gene is under the transcriptional control of said regulatoryelement.

However, in certain embodiments, the invention will not be limited touse with heterologous structural genes. The present disclosure, thus,additionally provides a method for sterol-mediated regulation ofexpression of a polypeptide in recombinant cell culture which includesthe steps of:

(a) preparing a first DNA segment which includes one or more sterolregulatory elements as described above;

(b) constructing a second DNA segment by positioning said first DNAsegment upstream from and proximal to a transcription initiation site ofa selected structural gene such that said structural gene may be broughtunder the transcriptional control of the sterol regulatory element ofsaid first DNA segment;

(c) transforming a host cell with said second DNA segment; and

culturing the transformed host cell in the presence of a sterol inamounts and for a time sufficient to suppress transcription of saidstructural gene.

In important and further embodiments, a method is provided fordetermining the ability of a candidate substance to activate thetranscription of DNA encoding the LDL receptor, which method comprises(a) providing a nucleic acid sequence containing the LDL receptor sterolregulatory element (SRE), a promoter and a reporter gene under thetranscriptional control of both of the SRE and the promoter which iscapable of conferring a detectable signal on a host cell, (b)transfecting said nucleic acid sequence into a host cell, (c) culturingthe cell, (d) contacting the cell culture with the candidate substance,and (e) assaying for the amount of signal produced by the cell culture.The greater the signal the greater the activating character of thecandidate. Transcriptionally activating candidate substances are thenevaluated further for potential as plasma cholesterol lowering drugsusing conventional techniques and animal models.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (parts A and B DNA Sequence of the Human LDL Receptor Promoter.The primary structure of a portion of the 5'-flanking region of thereceptor gene. Nucleotide +1 is assigned to the A of the ATG translationinitiation codon. Transcription initiation sites are indicated byasterisks. Two TATA-like sequences are underlined. Three imperfectdirect repeats of 6 bp are overlined with arrows in the promotersequence and aligned for homology at the bottom of the figure. Thesynthetic SRE 42 (Table III) differs from the 42-bp sequence shown hereby two nt (denoted by dots).

FIG. 2. Structure of human LDL receptor-CAT plasmids. Three fragments ofthe human LDL receptor promoter with a common 3' end at position -58were inserted into pSVO-CAT. The arrowhead indicates the region oftranscription initiation in the normal human LDL receptor gene (position-93 to -70). Black dots denote three imperfect direct repeats of 16 bp.

FIG. 3 (parts A and B). Sterol-mediated suppression of transfected andendogenous LDL receptor promoters in CHO cells. Panel A: Pooled CHOcells (150 to 600 colonies) co-transfected with pSV3-Neo and theindicated pLDLR-CAT plasmid were set up for experiments as described inExample I. Two different pools of cells transfected with pLDLR-CAT 234were studied. The cells were incubated for 20 hr in the absence orpresence of 10 ug/ml cholesterol plus 0.5 ug/ml 25-hydroxycholesterol,after which total RNA was isolated from 12 dishes of cells, and analiquot (20 ug) was used as a template in primer extension assays. Eachassay tube contained ³² P-labeled oligonucleotides specific for thetransfected neomycin-resistance gene (driven by the SV40 promoter) andthe CAT gene (driven by the LDL receptor promoter). The lanes on the farright show the primer extension products obtained when the CAT-specificor neomycin (Neo)-specific primers were used alone. The gel was exposedto X-ray film for 72 hr. For quantitation, the amounts of neomycin (a)and CAT (b) primer extension products were estimated by densitometry,and a ratio (b/a) of CAT-specific to neomycin-specific product wascalculated. Percent suppression was determined from this ratio. Panel B:The same RNA samples from Panel A were subjected to primer extensionanalysis using an oligonucleotide derived from exon 4 of the hamster LDLreceptor (LDLR) gene plus the neomycin (Neo)-specific oligonucleotide.The lane on the far right shows the result obtained with the hamster LDLreceptor primer alone. The black dots represent four products derivedfrom the endogenous LDL receptor mRNA. The longest extension product(575 nt, designated C) represents full-length extension to the mRNA capsite. The three shorter bands represent strong-stop sequencesencountered by the reverse transcriptase enzyme. Quantitation of "%suppression" was determined as described in Panel A. The gel was exposedto X-ray film for 48 hr. For Panels A and B, the positions to which DNAfragments of known size migrated are indicated on the right innucleotides (nt).

FIG. 4 (parts A and B Sterol-mediated suppression of transfectedpLDLR-CAT 1563 and endogenous LDL receptor promoter in CHO cells. Acloned line of CHO cells transfected with pLDLR-CAT 1563 was culturedaccording to the standard protocol. The cells were incubated for 20 hrwith the indicated amounts of cholesterol and 25-hydroxycholesterol(25-OH Chol.), after which total RNA was subjected to primer extensionanalysis as described in FIG. 3. In Panel A, the ³² P-labeledoligonucleotides were complementary to the mRNA produced by thetransfected pLDLR-CAT 1563 gene and the endogenous hamster TK gene. Thegel was exposed to X-ray film for 48 hr. In Panel B, the same RNAsamples were incubated with three ³² P-oligonucleotide primerscomplementary to the mRNAs derived from the endogenous hamster TK, LDLreceptor, and HMG CoA synthase genes. The black dots represent four mRNAproducts derived from the LDL receptor gene. The gel was exposed toX-ray film for 48 hr. In Panels A and B, the positions to whichradiolabeled markers migrated are indicated on the right.

FIG. 5. Quantification of sterol-mediated suppression of transfected andendogenous cholesterol-regulated genes in CHO cells. The amounts of theprimer extension products in FIG. 4 corresponding to mRNAs derived fromthe transfected LDL receptor-CAT 1563 gene, endogenous LDL receptor gene(575-nt product only), and endogenous HMG CoA synthase gene (bothproducts) were estimated by densitometry. The value for "100%expression" represents the amount of primer extension product observedin the absence of sterols.

FIG. 6. Time course of induct in of transfected pLDLR-CAT 1563 gene inCHO cells. A cloned line of CHO cells transfected with pLDLR-CAT 1563was cultured according to the standard protocol. The cells wereincubated for 20 hr with suppression medium containing 10 ug/mlcholesterol and 0.5 ug/ml 25-hydroxycholesterol and then switched toinduction medium lacking sterols for the indicated time period. Theremoval of sterols was staggered in such a way that all cells wereharvested at the same time of day 3 of cell growth. Total cellular RNAwas isolated and subjected to primer extension analysis witholigonucleotides specific for the transfected CAT gene mRNA and theendogenous hamster TK gene mRNA (Inset). The gel was exposed to X-rayfilm for 24 hr. For quantitation of "% maximum expression", a ratio ofthe relative amounts of the LDLR-CAT and TK primer extension productswas determined by densitometry. A value of 100% was assigned to theratio obtained after 48 hr in induction medium.

FIG. 7 (parts A and B Nucleotide sequences of normal and mutant LDLreceptor promoters. The sequence of a portion of the normal LDL receptorpromoter is shown at the top and numbered according to a convention inwhich the A of the ATG initiation codon is +1. Dots are placed above thesequence every 10 nt beginning at -80. Transcription initiation sitesare indicated by asterisks, and two TATA-like sequences are underlined.Three imperfect direct repeats of 16 nt are indicated by the arrowsbeneath the sequence. Below the sequence of the normal promoter areshown 15 overlapping mutations that were separately introduced into theDNA by site-directed oligonucleotide mutagenesis. The mutations arelabeled on the left according to the 10-bp sequence that was scrambled.The novel sequence that was introduced is shown in lower case lettersbelow the normal promoter sequence. Dashes indicate nucleotides that areidentical between the normal and mutant promoters.

FIG. 8. Expression and regulation of transfected pLDLR-CAT andpHSVTK-CAT genes in CHO cells. Plasmid pHSVTK-CAT was constructed fromplasmids pTK-CAT of Cato et al. (1986), EMBO J., 5:2237) and plasmid105/115 of McKnight and Kingsbury (1982), Science, 217:316). It containsHSVTK promoter sequences spanning base pairs -108 to +55 and has a BamHIlinker inserted at position -108. In the experiment, plasmid pLDLR-CAT234 or the indicated derivatives containing 10-bp scramble mutations(FIG. 7) were cotransfected with pHSVTK-CAT and pSV3-Neo into CHO cellsand assayed for expression and regulation by primer extension. Eachpooled cell line (300-600 independent colonies) was set up for assayaccording to the standard protocol described in Example I. Afterincubation for 20 hr in the absence (-) or presence (+) of 10 ug/mlcholesterol and 0.5 ug/ml 25-hydroxycholesterol, RNA was prepared andsubjected to primer extension analysis using ³² P-labeledoligonucleotides specific for the products of the two transfected CATgenes and for the endogenous hamster TK gene product. The gel wasexposed to X-ray film for 48 hr. For quantitation of "% suppression",the relative amounts of the transfected LDLR-CAT (b) and HSVTK-CAT (a)primer extension products were determined by densitometry, and a ratio(b/a) of the two was calculated. The sizes of the primer extensionproducts (right) were determined by comparison to the migration of DNAfragments of known molecular weight electrophoresed in adjacent lanes(not shown).

FIG. 9. Relative transcription activity of normal and mutant LDLreceptor promoters. Relative transcription activity is expressed as theratio (b/a) of the amounts of primer extension products corresponding tomRNAs derived from the transfected LDL receptor-CAT gene (b) and fromthe HSVTK-CAT gene (a) as shown in FIG. 7. A value of 1.0 (dashedhorizontal line) was assigned to the ratio comparing the normal LDLreceptor promoter (pLDLR-CAT 234) to the HSVTK-CAT promoter. The ratiosobtained from the 15 different scramble mutations (FIG. 7) and theirrelative locations in the LDL receptor promoter are indicated by theheight and width of the blocks, respectively, in the histogram. The datashown represent the average of 2 to 5 separate transfection experiments.A schematic of the normal LDL receptor promoter and its relevantlandmarks is shown at the bottom of the figure.

FIG. 10. Structure of plasmids containing LDL receptor promoter linkedto HSV TK-CAT gene. Different fragments of the human LDL receptorpromoter DNA were 25 linked to the HSV TK promoter at position -32(plasmids B-D) or -60 (plasmids F-H). The starting plasmid HSV TK 32-CAT(A) contains 32 bp upstream of the viral TK cap site and includes a TATAsequence as well as 55 bp to TK 5' untranslated sequences. The plasmidHSV TK 60-CAT (E) contains 60 bp upstream of the cap site of the viralTK gene and includes a TATA sequence and the first upstream regulatorysignal (GC box) of the viral promoter. The 5'-flanking sequences of theLDL receptor gene are denoted by the hatched line and are numberedaccording to FIG. 1. Three 16-bp imperfect direct repeats are indicatedby thick black arrows.

FIG. 11. (parts A and B Sterol-mediated regulation of HSV TK promotorcontaining synthetic LDL receptor SRE (42-mer). Top Panel: A synthetic42-bp fragment of DNA corresponding to sequences between -165 and -126of the human LDL receptor promoter (SRE 42) was inserted in varyingnumbers and orientations into a plasmid containing the HSV TK promoterlinked to CAT (Table II). Each 42-bp sequence contains two copies of animperfect =16 bp direct repeat denoted by heavy arrows. The viral TK-CATplasmid (plasmid I) contains a 10-bp BamHI linker (hatched areas)between positions -48 and -32 relative to the TK cap site. Bottom Panel:Plasmids I-N were transfected into CHO cells. Each resulting pooled cellline (300-600 colonies) was set up for experiments according to thestandard protocol. The cells were incubated for 20 hr in the absence orpresence of 10 ug/ml cholesterol and 0.5 ug/ml 25-hydroxycholesterol,after which 20 ug of total RNA was used as a template for primerextension analysis employing ³² P-labeled oligonucleotides specific forthe mRNAs of the transfected HSV TK-CAT gene product, the endogenoushamster TK gene product, and the endogenous HMG CoA synthase geneproduct. The gel was exposed to X-ray film for 72 hr. For quantitationof "% suppression", the relative amounts of the viral (b) and endogenous(a) TK primer extension products were determined by densitometry, and aratio (b/a) of the two was calculated. Only one of the two synthase mRNAproducts is shown. FIG. 12 (parts A and B. Structure of plasmidscontaining synthetic footprint 3 or repeat 3 sequences from LDL receptorpromoter inserted into HSV TK-Cat gene. Top Panel: Plasmids O through Rwere constructed using plasmid I (FIG. 4) as a starting vector intowhich two synthetic oligonucleotide sequences were inserted in bothorientations (see Table I). Plasmid I contains an HSV TK promoter inwhich a 10-bp BamHI linker has been substituted for sequences between-32 and -48. Plasmids Q and R contain two copies of one of the 16-bpdirect repeats found in the human LDL receptor promoter. The directrepeat sequences in plasmids Q and R are separated by a AGATCT linker(hatched regions). Bottom Panel: Plasmids O-R were transfected into CHOcells. Each resulting pooled cell line (300-600 colonies) was set up forexperiments according to the standard protocol. The cells were incubatedfor 20 hr in the absence or presence of 10 ug/ml cholesterol and 0.5ug/ml 25-hydroxy-cholesterol, after which total RNA was used as atemplate for primer extension analysis employing ³² P-labeledoligonucleotides specific for the HSV TK gene product, the endogenoushamster TK gene product, or the endogenous HMG CoA synthase geneproduct. The gels were exposed to X-ray film for 72 hr. For quantitationof "% suppression", the relative amounts of the transfected HSV TK (b)and endogenous (a) TK primer extension products were determined bydensitometry, and a ratio (b/a) of the two was calculated. Only one ofthe two synthase products is shown.

FIG. 13. Diagram demonstrating the construction of an expression vectorfor human growth hormone using an LDL receptor SRE promoter.

FIG. 14. Diagram demonstrating the construction of an expression vectorfor human tumor necrosis factor (TNF) using an LDL receptor SREpromoter.

FIG. 15. Diagram demonstrating the construction of an expression vectorfor human tissue plasminogen activator (t-PA) using an LDL receptor SREpromoter.

FIG. 16. (parts A and B) Sterol-mediated regulation of chimeric genescontaining the LDL receptor promoter upstream regulatory sequences inforward and reverse orientations. The 1507-base pair EcoRl/Alulrestriction fragment from the 5' end of the LDL receptor gene used toconstruct p1471 is shown at the top. Nucleotide positions -1471 to +36are numbered in relation to the transcription initiation site, indicatedby the arrow at +1. A putative TATA box is designated T/A. The tworegions that bind Spl in vitro (repeats 1 and 3) are shown, as is theSRE-I within repeat 2. The CAT coding sequence is denoted by thecross-hatched bar. To construct pZ, the region from -33 to -110 of p1471Was inverted by M13 mutagenesis (Zoller et al. (1984), DNA, 3:479-488).Relative transcription was determined by a primer extension assay of theamount of CAT mRNA in stably transfected CHO cells incubated for 24 hrin the absence or presence of sterols. LDL receptor promoter testplasmid expression was normalized to pTK-CAT-1 control plasmidexpression in each experiment. The data are expressed as a fraction ofthe level observed in cells transfected with p1471 and cultured in theabsence of sterols in the same experiment. Each value represents theaverage ±S.E.M. of four experiments. The gel was dried and exposed toX-ray film at -70° C. with an intensifying screen for 75 h. Foldinduction was calculated as the ratio of normalized test plasmidexpression in the absence of sterols to that in the presence of sterolsfor each experiment. w.t., wild-type; TK, thymidine kinase; LDLR, LDLreceptor.

FIG. 17 (parts A and B). Nucleotide sequences and relative transcriptionrates of mutant LDL receptor promoters. The horizontal axis shows thesequence of repeat 2 of the human LDL receptor promoter. The eightnucleotides comprising the core SRE-1 sequence in repeat 2 areunderlined. Arrows indicate point mutations, designated a-r. Panel A,the height of each bar represents the amount of LDL receptor-CAT mRNAproduced from each plasmid relative to the amount of mRNA produced bythe internal control (pTK-CAT-1) in each assay. The amount of mRNAproduced by the wild-type LDL receptor promoter was arbitrarily assigneda value of 1.0. The data was derived from densitometric scans ofautoadiograms as shown in FIG. 18. The data represent the relativetranscription observed when cells were cultured in the absence (openbar) or presence (shaded bar) of sterols as described under"Experimental Procedures." The standard error of the mean (S.E.M.) ofdata for the given number of experiments (n with each mutant is plottedat the top of each bar. Panel B, fold induction of transcription fromplasmids driven by the wild-type (w.t.) and mutant (a-r) LDL receptorpromoters in transfected CHO cells grown in the presence and absence ofsterols. Fold induction was calculated as described in the legend toFIG. 16 and plotted on the vertical axis. Each plasmid was transfectedtwice, once using Protocol 1 and once using Protocol 2. The datapresented in Panels A and B represent the mean of 3023 experiments fromboth transfections for various plasmids. TK, thymidine kinase; LDLR, LDLreceptor.

FIG. 18 (parts A-H). Expression of mutant LDL receptor promoters intransfected CHO cells incubated in the absence and presence of sterols.Each of the mutant LDL receptor promoters shown in FIG. 17 was fused tothe CAT coding sequence and transfected into CHO cells together with acontrol plasmid containing the HSV thymidine kinase promoter fused tothe CAT coding sequence (pTK-CAT-1). Transfections in Panels A-D and Hwere carried out according to Protocol 1; transfections in Panels E-Gwere carried out according to Protocol 2. Stably transfected colonieswere selected, RNA was harvested from pooled colonies after growth inthe absence and presence of sterols, and primer extension assays wereperformed. After drying, the gels shown in Panels A, B, C, D, E, F, GAND H were exposed to X-ray film at -70° C. with an intensifying screenfor 33, 48, 41, 138, 120, 99, 99 and 138 hr, respectively. TK, thymidinekinase; LDLR, LDL receptor.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Animal cells regulate their cholesterol content through the integrationof two pathways that govern the supply of exogenous and endogenouscholesterol. Both pathways are controlled by end-product repression.Preferentially, they obtain cholesterol through the receptor-mediatedendocytosis and lysosomal hydrolysis of plasma low density lipoprotein.However, when cells are depleted of cholesterol, they synthesize largeamounts of mRNA for the low density lipoprotein (LDL) receptor, whichfacilitates the uptake of exogenous cholesterol by receptor-mediatedendocytosis. The cells also increase their endogenous cholesterolproduction by increasing the amount of mRNA for two sequential enzymesin de novo cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzymeA (HMG CoA) synthase and HMG CoA reductase. When cholesterol builds upwithin the cell, all three of these mRNAs are strongly suppressed, anaction that limits both the uptake and synthesis of cholesterol.

The present invention embodies the realization that the precise geneticelements which are responsible for this sterol-induced feedbackrepression of LDL receptor production can be isolated away from the LDLreceptor gene and employed to confer sterol regulatory capability toheterologous genes. Moreover, additional control elements containedwithin the sterol regulatory sequences have been found to confertranscription promotion capability without conferring sterol regulationper se.

The novel nucleic acid sequences of the present invention comprise (1)sequences which provide sterol regulatory capability to heterologousstructural genes with or without a positive promotion of transcription(SREs); or (2) sequences which provide a positive promotion oftranscription without providing a sterol regulatory capability.

It is now clear that these positive and sterol-mediated control elementsreside on separate, but structurally similar, DNA sequences 16nucleotides in length. Due to their relatively short length, thesesequences may be, and have been, routinely synthesized using DNAsynthesizers, thus obviating a need for isolation of the sequences fromnatural sources.

The inventors have discovered and characterized discreet sequenceregions which exist upstream of the sterol mediated structural genes.Surprisingly, the minimal sequence requirement, or "core" region, for loconferring SRE regulation capability has been determined to be localizedto an 8 to 10 base pair sequence region. This "core SRE region" actssomewhat like an enhancer element: in the absence of sterols, the SREserves to simulate transcription rate several fold, whereas in thepresence of sterols, it loses this capability.

The present invention contemplates that the basic 8 to 10 nucleotidecore SRE region can be employed in connection with any selectedheterologous or even homologous structural gene. This basic unit hasbeen characterized from an analysis of upstream control regions ofsterol-mediated structural genes. Thus, it will typically be preferredto employ longer sequence stretches that correspond to the identifiedregions. This is because although the core region is all that isultimately required, it is believed that particular advantages accrue,in terms of sterol regulation and level of induction achieved in theabsence of sterol repression, where one employs sequences whichcorrespond to the natural SRE control regions over longer regions, e.g.,up to around 25 or so nucleotides.

These "extended" regions discovered by the inventors to confersterol-mediated regulation in the case of the sterol-mediated structuralgenes are as follows: ##STR1##

The inventors have conducted numerous studies to delineate andcharacterize with some degree of particularity the core SRE region. Asnoted, these studies have led the inventors to conclude that at least an8 nucleotide region is required to confer an SRE function. This mostbasic element can be set forth as follows:

    5'C-A-C-M-S-Y-A-C 3';

wherein:

Y=C or T

S=C or G

M=C or A;

or the complement of such a sequence.

However, from the inventors studies, it is contemplated that theincorporation of two additional nucleotides, either G or C, to the 5'end of this 8 nucleotides stretch will provide additional advantages inaccordance with the invention. Thus, in these embodiments, the SRE maybe defined more particularly as follows:

    5'-A-T-C-A-C-M-S-Y-A-C-3'

wherein:

Y=C or T

S=C or G

M=C or A;

or the complement of such a sequence.

Also from the inventors studies, it was found that additional nucleotideregions positioned 5' and/or 3' with respect to the central core mayprovide still further advantages. In these embodiments, the SRE may bedefined as follows:

(a) 5'-M-M-M-A-T-C-A-C-M-S-Y-A-C-3'

wherein:

Y=C or T

S=C or G

M=C or A; or

(b) 5'-M-M-M-A-T-C-A-C-M-S-Y-A-C-K-K-M-3'

wherein:

K=T or G

Y=C or T

S=C or G

M=C or A; or the complement of such a sequence.

Positive control sequences identified by the inventors, which do notprovide an SRE capability, are preferably defined by the sequences:

(a) 5'-A-A-A-C-T-C-C-T-C-C-T-C-T-T-G-C-3';

(b) 5'-A-A-A-C-T-C-C-T-C-C-C-C-C-T-G-C-3';

(c) 5'-G-C-A-A-G-A-G-G-A-G-G-A-G-T-T-T-3'; and

(d) 5'-G-C-A-G-G-G-G-G-A-G-G-A-G-T-T-T-3'.

As with the sterol regulatory elements, it will be appreciated thatpositive promoter segments (c) and (d) represent the opposite strandsequence of promoter segments (a) and (b).

Thus, both a positive and/or a sterol-mediated control is provided byselecting one or more segments from classes of the foregoing controlsequences and locating such sequences upstream from and proximal to aheterologous transcription initiation site.

The novel control sequences of the present invention, whether positive,sterol-mediated, or both, may be even more advantageously employed inthe form of multiple units, in numerous various combinations andorganizations, in forward or reverse orientations, and the like.Moreover, in the context of multiple unit embodiments and/or inembodiments which incorporate both positive and negative control units,there is no requirement that such units be arranged in an adjacenthead-to-head or head-to-tail construction in that the improvedregulation capability of such multiple units is conferred virtuallyindependent of the location of such multiple sequences with respect toeach other. Moreover, there is no requirement that each unit comprisethe same positive or negative element. All that is required is that suchsequences be located upstream of and sufficiently proximal to atranscription initiation site. However, in a preferred aspect of theimproved multiple unit embodiment, the control sequences are locatedwithin from 0-20 nucleotides of each other.

When employed in the context of heterologous structural genes, theprecise location of the control sequences of the invention with respectto transcription initiation site is not particularly crucial. Forexample, some benefit will generally be obtained when such controlsequences are located up to about 300 nucleotides or more from atranscription initiation site. However, in more preferred embodiments,control sequences are located within 150 nucleotides of such a site.Still more benefit is obtained when the sequences are located within 100nucleotides of initiation. Moreover, control sequences are mostadvantageously employed when disposed within 50 nucleotides oftranscription initiation. Thus, in general, the closer the controlsequences are to transcription initiation, the more pronounced andeffective the control obtained.

Therefore, to employ the foregoing regulatory elements in the context ofheterologous genes, one simply obtains the structural gene and locatesone or more of such control sequences upstream of a transcriptioninitiation site. Additionally, as is known in the art, it is generallydesirable to include TATA-box sequences upstream of and proximal to atranscription initiation site of the heterologous structural gene. Suchsequences may be synthesized and inserted in the same manner as thenovel control sequences. Alternatively, one may desire to simply employthe TATA sequences normally associated with the heterologous gene. Inany event, TATA sequences are most desirably located between about 20and 30 nucleotides upstream of transcription initiation.

Numerous methods are known in the art for precisely locating selectedsequences at selected points within larger sequences. Most conveniently,the desired control sequence or sequences, or combinations of sequences,are synthesized and restriction site linker fragments added to thecontrol sequence termini. This allows for ready insertion of controlsequences into compatible restriction sites within upstream regions.Alternatively, synthesized control sequences may be ligated directly toselected regions. Moreover, site specific mutagenesis may be employed tofashion restriction sites into which control sequences may be insertedin the case where no convenient restriction sites are found at a desiredinsertion site.

As noted, it is believed that the control sequences of the presentinvention may be beneficially employed in the context of anyheterologous structural gene, with or without additional homologous orheterologous control or promotion sequences. The following table, TableI, lists a number of known defined structural genes, along withdescriptive references, which may be employed in the context of thecontrol sequences of the present invention. It should, however, beappreciated that this table is in no way intended to be an exhaustive orall-inclusive listing, and it is included herein for the convenience ofthe reader. For a more extensive listing, one may wish to refer toBeaudet (1985), Am. J. Hum. Gen., 37:386-406.

                                      TABLE I    __________________________________________________________________________    Selected Cloned Structural Genes    Gene            Clone Type*  Reference    __________________________________________________________________________    activin         porcine-cDNA Mason AJ, Nat, 318:659, 1985    adenosine deaminase                    h-cDNA       Wiginton DA, PNAS, 80:7481, 1983    angiotensinogen I                    r-cDNA       Ohkubo H, PNAS, 80:2196, 1983                    r-gDNA       Tanaka T, JBC, 259:8063, 1984    antithrombin III                    h-cDNA       Bock SC, NAR 10:8113, 1982                    h-cDNA and gDNA                                 Prochownik EV, JBC, 258:8389, 1983    antitrypsin, alpha I                    h-cDNA       Kurachi K, PNAS, 78:6826, 1981                    h-gDNA       Leicht M, Nat, 297:655, 1982                    RFLP         Cox DW, AJHG, 36:134S, 1984    apolipoprotein A-I                    h-cDNA, h-gDNA                                 Shoulders CC, NAR, 10:4873, 1982                    RFLP         Karathanasis SK, Nat, 301:718, 1983                    h-gDNA       Kranthanasis SK, PNAS, 80:6147, 1983    apolipoprotein A-II                    h-cDNA       Sharpe CR, NAR, 12:3917, 1984                    Chr          Sakaguchi AY, AJHG, 36:207S, 1984                    h-cDNA       Knott TJ, BBRC, 120:734, 1984    apolipoprotein C-1                    h-cDNA       Knott TJ, NAR, 12:3909, 1984    apolipoprotein C-II                    h-cDNA       Jackson CL, PNAS, 81:2945, 1984                    h-cDNA       Mykelbost O, JBC, 259:4401, 1984                    h-cDNA       Fojo SS, PNAS, 81:6354, 1984                    RFLP         Humphries SE, C Gen, 26:389, 1984    apolipoprotein C-III                    h-cDNA and gDNA                                 Karanthanasis SK, Nat, 304:371, 1983                    h-cDNA       Sharpe CR, NAR, 12:3917, 1984    apolipoprotein E                    h-cDNA       Breslow JL, JBC, 257:14639, 1982    atrial natriuretic factor                    h-cDNA       Oikawa S, Nat, 309:724, 1984                    h-cDNA       Nakayama K, Nat, 310:699, 1984                    h-cDNA       Zivin RA, PNAS, 81:6325, 1984                    h-gDNA       Seidman CE, Sci, 226:1206, 194                    h-gDNA       Nemer M, Nat, 312:654, 1984                    h-gDNA       Greenberg BI, Nat, 312:656, 1984    chorionic gonadotropin,                    h-cDNA       Fiddes JC, Nat, 281:351, 1981    alpha chain     RFLP         Boethby M, JBC, 256:5121, 1981    chorionic gonadotropin,                    h-cDNA       Fiddes JC, Nat, 286:684, 1980    beta chain      h-gDNA       Boorstein WR, Nat, 300:419, 1982                    h-gDNA       Talmadge K, Nat, 307,37, 1984    chymosin, pro (rennin)                    bovine-cDNA  Harris TJR, NAR, 10:2177, 1982    complement, factor B                    h-cDNA       Woods DE, PNAS, 79:5661, 1982                    h-cDNA and gDNA                                 Duncan R, PNAS, 80:4464, 1983    complement C2   h-cDNA       Bentley DR, PNAS, 81:1212, 1984                    h-gDNA (C2, C4, and B)                                 Carroll MC, Nat, 307:237, 1984    complement C3   m-cDNA       Domdey H, PNAS, 79:7619, 1983                    h-gDNA       Whitehead AS, PNAS, 79:5021, 1982    complement C4   h-cDNA and gDNA                                 Carroll MC, PNAS, 80:264, 1983                    h-cDNA       Whitehead AS, PNAS, 80:5387, 1983    complement C9   h-cDNA       DiScipio RC, PNAS, 81:7298, 1984    corticotropin releasing                    sheep-cDNA   Furutani Y, Nat, 301:537, 1983    factor          h-gDNA       Shibahara S, EMBO J, 2:775, 1983    epidermal growth factor                    m-cDNA       Gray A, Nat, 303:722, 1983                    m-cDNA       Scott J, Sci, 221:236, 1983                    h-gDNA       Brissenden JE, Nat, 310:781, 1984    epidermal growth factor                    h-cDNA and Chr                                 Lan CR, Sci, 224:843, 1984    receptor, oncogene    c-erb B    epoxide dehydratase                    r-cDNA       Gonzalez FJ, JBC, 256:4697, 1981    erythropoietin  h-cDNA       Lee-Huang S, PNAS, 81:2708, 1984    esterase inhibitor, Cl                    h-cDNA,      Stanley KK, EMBO J, 3:1429, 1984    factor VIII     h-cDNA and gDNA                                 Gitschier J, Nat, 312:326, 1984                    h-cDNA       Toole JJ, Nat, 312:342, 1984    factor IX, Christmas factor                    h-cDNA       Kutachi K, PNAS, 79:6461, 1982                    h-cDNA       Choo KH, Nat, 299:178, 1982                    RFLP         Camerino G, PNAS, 81:498, 1984                    h-gDNA       Anson DS, EMBO J, 3:1053, 1984    factor X        h-cDNA       Leytus SP, PNAS, 81:3699, 1984    fibrinogen A alpha,                    h-cDNA       Kant JA, PNAS, 80:3953, 1983    B beta, gamma   h-gDNA (gamma)                                 Fornace AJ, Sci, 224:161, 1984                    h-cDNA (alpha gamma)                                 Imam AMA, NAR, 11:7427, 1983                    h-gDNA (gamma)                                 Fornace AJ, JBC, 259:12826, 1984    gatrin releasing peptide                    h-cDNA       Spindel ER, PNAS, 81:5699, 1984    glucagon, prepro                    hamster-cDNA Bell GI, Nat, 302:716, 1983                    h-gDNA       Bell GI, Nat, 304,368, 1983    growth hormone  h-cDNA       Martial JA, Sci, 205:602, 1979                    h-gDNA       DeNoto FM, NAR, 9:3719, 1981                    GH-like gene Owerbach D, Sci, 209:289, 1980    growth hormone RF,                    h-cDNA       Gubler V, PNAS, 80:4311, 1983    somatocrinin    h-cDNA       Mayo KE, Nat, 306:86, 1983    hemopexin       h-cDNA       Stanley KK, EMBO J, 3:1429, 1984    inhibin         porcine-cDNA Mason AJ, Nat, 318:659, 1985    insulin, prepro h-gDNA       Ullrich A, Sci, 209:612, 1980    insulin-like growth factor I                    h-cDNA       Jansen M, Nat, 306:609, 1983                    h-cDNA       Bell GI, Nat, 310:775, 1984                    Chr          Brissenden JE, Nat, 310:781, 1984    insulin-like growth factor II                    h-cDNA       Bell GI, Nat, 310:775, 1984                    h-gDNA       Dull TJ, Nat, 310:777, 1984                    Chr          Brissenden JE, Nat, 310:781, 1984    interferon, alpha                    h-cDNA       Maeda S, PNAS, 77:7010, 1980    (leukocyte), multiple                    h-cDNA (8 distinct)                                 Goeddel DV, Nat, 290:20, 1981                    h-gDNA       Lawn RM, PNAS, 78:5435, 1981                    h-gDNA       Todokoro K, EMBO J, 3:1809, 1984                    h-gDNA       Torczynski RM, PNAS, 81:6451, 1984    interferon, beta (fibroblast)                    h-cDNA       Taniguchi T, Gene, 10:11, 1980                    h-gDNA       Lawn RM, NAR, 9:1045, 1981                    h-gDNA (related)                                 Sehgal PB, PNAS, 80:3632, 1983                    h-gDNA (related)                                 Sagar AD, Sci, 223:1312, 1984    interferon, gamma (immune)                    h-cDNA       Gray PW, Nat, 295:503, 1982                    h-gDNA       Gray PW, Nat, 298:859, 1982    interleukin-1   m-cDNA       Lomedico PT, Nat, 312:458, 1984    interleukin-2, T-cell                    h-cDNA       Devos R, NAR, 11:4307, 1983    growth factor   h-cDNA       Taniguchi T, Nat, 302:305, 1983                    h-gDNA       Hollbrook NJ, PNAS, 81:1634, 1984                    Chr          Siegel LJ, Sci, 223:175, 1984    interleukin-3   m-cDNA       Fung MC, Nat, 307:233, 1984    kininogen, two forms                    bovine-cDNA  Nawa H, PNAS, 80:90, 1983                    bovine-cDNA and gDNA                                 Kitamura N, Nat, 305:545, 1983    luteinizing hormone,                    h-gDNA and Chr                                 Talmadge K, Nat, 207:37, 1984    beta subunit    luteinizing hormone                    h-cDNA and gDNA                                 Seeburg PH, Nat, 311:666, 1984    releasing hormone    lymphotoxin     h-cDNA and gDNA                                 Gray PW, Nat, 312:721, 1984    mast cell growth factor                    m-cDNA       Yokoya T, PNAS, 81:1070, 1984    nerve growth factor, beta subunit                    m-cDNA       Scott J, Nat, 302:538, 1983                    h-gDNA       Ullrich A, Nat, 303:821, 1983                    Chr          Franke C, Sci, 222:1248, 1983    oncogene, c-sis, PGDF chain A                    h-gDNA       Dalla-Favera R, Nat, 295:31, 1981                    h-cDNA       Clarke MF, Nat, 208:464, 1984    pancreatic polypeptide and                    h-cDNA       Boel E, EMBO J, 3:909, 1984    icosapeptide    parathyroid hormone, prepro                    h-cDNA       Hendy GN, PNAS, 78:7365, 1981                    h-gDNA       Vasicek TJ, PNAS, 80:2127, 1983    plasminogen     h-cDNA and gDNA                                 Malinowski DP, Fed P, 42:1761, 1983    plasminogen activator                    h-cDNA       Edlund T, PNAS, 80:349, 1983                    h-cDNA       Pennica D, Nat, 301:214, 1983                    h-gDNA       Ny T, PNAS, 81:5355, 1984    prolactin       h-cDNA       Cook NE, JBC, 256:4007, 1981                    r-gDNA       Cooke NE, Nat, 297:603, 1982    proopiomelanocortin                    h-cDNA       DeBold CR, Sci, 220:721, 1983                    h-gDNA       Cochet M, Nat, 297:335, 1982    protein C       h-cDNA       Foster D, PNAS, 81:4766, 1984    prothrombin     bovine-cDNA  MacGillivray RTA, PNAS, 77:5153, 1980    relaxin         h-gDNA       Hudson P, Nat, 301:628, 1983                    h-cDNA (2 genes)                                 Hudson P, EMBO J, 3:2333, 1984                    Chr          Crawford RJ, EMBO J, 3:2341, 1984    renin, prepro   h-cDNA       Imai T, PNAS, 80:7405, 1983                    h-gdNA       Hobart PM, PNAS 81:5026, 1984                    h-gDNA       Miyazaki H, PNAS, 81:5999, 1984                    Chr          Chirgwin JM, SCMG, 10:415, 1984    somatostatin    h-cDNA       Shen IP, PNAS 79:4575, 1982                    h-gdNA and Ri-IP                                 Naylot SI, PNAS, 80:2686, 1983    tachykinin, prepro,                    bovine-cDNA  Nawa H, Nat, 306:32, 1983    substances P & K                    bovine-gDNA  Nawa H, Nat, 312:729, 1984    urokinase       h-cDNA       Verde P, PNAS, 81:4727, 1984    vasoactive intestinal                    h-cDNA       Itoh N, Nat, 304:547, 1983    peptide, prepro    vasopressin     r-cDNA       Schmale H, EMBO J, 2:763, 1983    __________________________________________________________________________     *cDNA . . . complementary DNA     Chr . . . chromosome     gDNA . . . genomic DNA     RFLP . . . restriction fragment polymorphism     hhuman     mmouse     rrat

With respect to the novel LDL receptor-stimulating drug screeningmethod, the method as provided herein preferably employs a reporter genethat confers on its recombinant hosts a readily detectable phenotypethat emerges only under the control of the LDL receptor SRE. Generallyreporter genes encode a polypeptide not otherwise produced by the hostcell which is detectable by in situ analysis of the cell culture, e.g.,by the direct fluorometric, radioisotopic or spectrophotometric analysisof the cell culture without the need to remove the cells for signalanalysis from the culture chamber in which they are contained.Preferably the gene encodes an enzyme which produces colorimetric orfluorometric change in the host cell which is detectable by in situanalysis and which is a quantitative or semi-quantitative function oftranscriptional activation. Exemplary enzymes include esterases,phosphatases, proteases (tissue plasminogen activator or urokinase) andother enzymes capable of being detected by activity which generates achromophore or fluorophore as will be known to those skilled in the art.

A preferred example is E. coli beta-galactosidase. This enzyme producesa color change upon cleavage of the indigogenic substrateindolyl-B-D-galactoside by cells bearing beta-galactosidase (see, e.g.,Goring et al., Science, 235:456-458 1987) and Price et al., Proc. Natl.Acad. Sci. USA, 84:156-160 (1987)). Thus, this enzyme facilitatesautomatic plate reader analysis of SRE-mediated expression directly inmicrotiter wells containing transformants treated with candidateactivators. Also, since the endogenous beta-galactosidase activity inmammalian cells ordinarily is quite low, the analytic screening systemusing B-galactosidase is not hampered by host cell background.

Another class of reporter genes which confer detectable characteristicson a host cell are those which encode polypeptides, generally enzymes,which render their transformants resistant against toxins, e.g., the neogene which protects host cells against toxic levels of the antibioticG418; a gene encoding dihydrofolate reductase, which confers resistanceto methotrexate, or the chloramphenicol acetyltransferase (CAT) gene(Osborne et al., Cell, 42:203-212 (1985)). Genes of this class are notpreferred since the phenotype (resistance) does not provide a convenientor rapid quantitative output. Resistance to antibiotic or toxin requiresdays of culture to confirm, or complex assay procedures if other than abiological determination is to be made.

Other genes for use in the screening assay herein are those capable oftransforming hosts to express unique cell surface antigens, e.g., viralenv proteins such as HIV gp120 or herpes gD, which are readilydetectable by immunoassays. However, antigenic reporters are notpreferred because, unlike enzymes, they are not catalytic and thus donot amplify their signals.

The polypeptide products of the reporter gene are secreted,intracellular or, as noted above, membrane bound polypeptides. If thepolypeptide is not ordinarily secreted it is fused to a heterologoussignal sequence for processing and secretion. In other circumstances thesignal is modified in order to remove sequences that interdictsecretion. For example, the herpes gD coat protein has been modified bysite directed deletion of its transmembrane binding domain, therebyfacilitating its secretion (EP 139,417A). This truncated form of theherpes gD protein is detectable in the culture medium by conventionalimmunoassays. Preferably, however, the products of the reporter gene arelodged in the intracellular or membrane compartments. Then they can befixed to the culture container, e.g. microtiter wells, in which they aregrown, followed by addition of a detectable signal generating substancesuch as a chromogenic substrate for reporter enzymes.

In general, an SRE is employed to control transcription and henceinfluence expression of the reporter gene. The process which in itsentirety leads to enhanced transcriptional promotion is termed"activation". The mechanism by which a successful candidate is acting isnot material in any case since the objective is to upregulate the LDLreceptor by whatever means will function to do so. While use of theentire LDL receptor promoter, including the SRE, will most closely modelthe therapeutic target, the SRE is optionally combined with more potentpromoters, e.g., the TK or SV40 early promoter described in the Examplesinfra in order to increase the sensitivity of the screening assay.

The SRE-containing promoter, whether a hybrid or the native LDL receptorpromoter, is ligated to DNA encoding the reporter gene by conventionalmethods. The SRE is obtained by in vitro synthesis or recovered fromgenomic DNA. It is ligated upstream of the start codon of the reportergene. The SRE-containing promoter also will contain an AT-rich region(TATA box) located between the SRE and the reporter gene start codon.The region 3' to the coding sequence for the reporter gene will containa transcription termination and polyadenylation site, for example thehepatitis B polyA site. The promoter and reporter gene are inserted intoa replicable vector and transfected into a cloning host such as E. coli,the host cultured and the replicated vector recovered in order toprepare sufficient quantities of the construction for later transfectioninto a suitable eukaryotic host.

The host cells used in the screening assay herein generally aremammalian cells, and preferably are permanent cell lines. Cell linesshould be stable and relatively easy to grow in large scale culture.Also, they should contain as little native background as possibleconsidering the nature of the reporter polypeptide. Examples include theHep G2, VERO, HeLa, CHO, W138, BHK, COS-7, and MDCK cell lines. TheSRE-containing containing vector is cotransfected into the desired host,stable transformants selected and, optionally, the reporter gene and itscontrolling SRE-containing promoter are amplified in order to increasethe screening assay sensitivity. This is accomplished in conventionalfashion by cotransforming the host with the reporter gene and aselectable marker gene such as DHFR (for DHFR minus host cells such asCHO) or DHFR and neo for other hosts, followed by the application of aselection agent.

The screening assay typically is conducted by growing the SREtransformants to confluency in microtiter wells, adding serial molarproportions of cholesterol and/or other sterols that suppress the SRE,and candidate to a series of wells, and the signal level determinedafter an incubation period that is sufficient to demonstratesterol-mediated repression of signal expression in controls incubatedsolely with 10 micrograms cholesterol/ml and 0.5 micrograms25-hydroxycholesterol/ml. The wells containing varying proportions ofcandidate are then evaluated for signal activation. Candidates thatdemonstrate dose related enhancement of reporter gene transcription orexpression are then selected for further evaluation as clinicaltherapeutic agents. The stimulation of transcription may be observed inthe absence of added sterols, in which case the candidate compound mightbe a positive stimulation of the SRE. Alternatively, the candidatecompound might only give a stimulation in the presence of sterols, whichwould indicate that it functions to oppose the sterol-mediatedsuppression of the SRE. Candidate compounds of either class might beuseful therapeutic agents that would stimulate production of LDLreceptors and thereby lower blood cholesterol in patients.

It should be understood that the screening method herein is usefulnotwithstanding that effective candidates may not be found, since itwould be a practical utility to know that SRE activators do not exist.The invention consists of providing a method for screening for suchcandidates, not in finding them. While initial candidate agents might besterol derivatives, at this time it is unknown which sterol derivatives,if any, will be efficacious.

EXAMPLE I Expression and Regulation of Human LDL Receptor Promoter-CATGenes

From a consideration of the nucleotide sequence of the 5' region of thehuman LDL receptor gene (see FIG. 1), three 16 base sequences wereobserved whose sequences appeared to be at least partially conservedwith respect to each other. It was initially hypothesized by the presentinventors that these sequences, alone or in combination with each otheror in association with flanking sequences may function to provide sterolregulation to the LDL receptor gene.

Various different experimental approaches have been employed by thepresent inventors to demonstrate that these 5'-flanking sequencescontain transcription signals that confer both positive and negativeregulation. In one approach, hybrid genes have been constructed from LDLreceptor 5'-flanking sequences and those of the herpes simplex virusthymidine kinase (HSV TK) gene (see Example II). These studies showedthat the two more proximal direct repeats (repeats 2 and 3) harbored aregulatory sequence that responded in a negative manner to the level ofsterols in the culture medium. This sequence is referred to by thepresent inventors as the Sterol Regulatory Element (SRE) of the LDLreceptor gene.

The present example reflects experiments conducted to display generallythe positive regulatory capability of the 5' regions. In this regard,fusion genes constructed between a marker gene and up to 6500 bp of5'-flanking DNA of the LDL receptor gene identified a 177-bp fragment ofthe receptor gene that contained signals for both positive expressionand negative regulation by sterols. The sequences responsible forpositive expression were further delineated by analyzing a series of 15mutations in the 177-bp promoter fragment, in which overlapping 10-bpsegments were scrambled by site-directed mutagenesis. The results ofthese studies indicate that each of the three direct repeats as well asone of the TATA sequences in the receptor promoter are preferred for LDLreceptor mRNA expression. Comparison of the direct repeat sequences witha newly derived consensus sequence recognized by the eukaryotictranscription factor Sp1 reveals a sufficient degree of homology tosuggest to the present inventors that this protein may play a role inthe expression of the LDL receptor gene.

For the experiments which follow, a series of three plasmids wereconstructed in which 5'-flanking sequences of the LDL receptor gene werefused to the bacterial CAT gene (chloramphericol acetyl transferase)(see FIG. 2). E. coli cells harboring plasmid pLDL-CAT 234 have beendeposited with the ATCC on Mar. 30, 1987, and accorded ATCC designation67375.

Abbreviations used bp, base pairs; CAT, chloroamphenicolacetyltransferase; CHO, Chinese hamster ovary; HMG CoA,3-hydroxy-3-methylglutaryl CoA; HSVTK, herpes simplex virus thymidinekinase; kb, kilobase(s); LDL, low density lipoprotein; nt, nucleotide;SRE, sterol regulatory element; TE buffer, 10 mM Tris-chloride and 1 mMEDTA at pH 8; TK, thymidine kinase.

SELECTED MATERIALS AND METHODS EMPLOYED Materials

[gamma-³² P]ATP (>5000 Ci/mmole) was obtained from ICN. Enzymes used inplasmid constructions were obtained from New England Biolabs andBoehringer Mannheim Biochemicals. Reverse transcriptase was purchasedfrom Life Sciences (Cat. No. AMV 007). G418 sulfate (Geneticin) waspurchased from GIBCO Laboratories. Plasmid pSV3-Neo, which contains abacterial gene that confers resistance to G418 (Southern et al. (1982),J. Mol. Appl. Gen., 1:327), was obtained from Bethesda ResearchLaboratories. Cholesterol and 25-hydroxycholesterol were purchased fromAlltech Associates and Steraloids, Inc., respectively. Plasmid pSVO-CAT(Gorman et al. (1982), Moll Cell. Biol., 2:1044) was kindly provided byDr. Bruce Howard. Newborn calf lipoprotein-deficient serum (d>215 g/ml)was prepared by ultracentrifugation (Goldstein et al. (1983), Meth.Enzymol., 98:241). Oligonucleotides were synthesized on an AppliedBiosystems Model 380A DNA synthesizer.

Plasmid Constructions

1) LDL Receptor Promoter-CAT Genes. A series of plasmids was constructedby standard techniques of genetic engineering (Maniatis et al. (1982),Molecular Cloning: A Laboratory Manual, Cold spring Harbor Laboratory,Press, N.Y., pp 1-545). These plasmids contained fragments of the LDLreceptor promoter extending for various distances in the 5' directionand terminating at position -58 linked to the bacterial gene encodingchloramphenicol acetyl transferase (CAT). The LDL receptor fragmentswere inserted into the unique HindIII site of pSVO-CAT, a recombinantplasmid that contains the beta-lactamase gene, the origin of replicationfrom pBR322, and the coding sequence for CAT (Gorman et al., supra). Allof the cloning junctions in the resulting series of LDL receptor-CATgenes were verified by DNA sequence analysis and restrictionendonuclease mapping.

2) Scramble Mutations in LDL Receptor Promoter-Cat Genes. To constructthe series of 15 promoter mutations diagramed in FIG. 7, a 1.8-kilobase(kb) EcoRI-PstI fragment containing the LDL receptor promoter wasexcised from plasmid pLDLR-CAT 234 and cloned into the bacteriophage M13mp19 vector (Messing (1983), Meth. Enzymol., 101:20). Site specificmutagenesis was performed on single stranded M13 recombinant DNA usingthe single primer method of Zoller and Smith (Zoller et al. (1984), DNA,3:47a). Mutagenic oligonucleotides of 40-42 bases in length wereemployed in which the 10 base sequence to be scrambled was located inthe center of the oligonucleotide. To facilitate unambiguousidentification of a given mutant, each of the introduced 10 basesequences contained a novel NsiI and/or SphI site. After annealing andextension with the large fragment of DNA polymerase I in the presence ofbacteriophage T4 DNA ligase, the double-stranded M13 DNA was transformedinto E. coli TGl cells. Plaques containing the desired mutation wereidentified by hybridization with the radiolabeled mutagenicoligonucleotide, subjected to one round of plaque purification, and thensequenced by the methods of Sanger, et al. (1980), J. Mol. Biol.,143:161). The EcoRI-PstI fragment containing the mutation was excisedfrom the M13 clone after conversion of the single stranded DNA intodouble stranded DNA by primer extension (Maniatis et al., supra) andthen recloned into the pSVO-CAT backbone. The resulting plasmid wascharacterized by restriction mapping with NsiI or SphI and DNAsequencing and then assigned a name according to the 10-bp sequencescrambled; e.g., pLDLR-CAT -228/-219 harbors an LDL receptor promoterfragment extending from -234 to -58 (FIG. 2) in which the normal 10-bpsequence between -228 and -219 (GGGTTAAAAG) has been replaced withATATGCATGC (FIG. 7).

DNA Transfection and G418 Selection

All cells were grown in monolayer culture at 37˜C in an atmosphere of5%-7% Co₂. CHO-K1 cells were maintained in medium A (Ham's F12 mediumcontaining 17 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid atpH 7.4, 21 mM glutamine, 100 U/ml penicillin, and 100 ug/mlstreptomycin) supplemented with 10% (v/v) fetal calf serum. Cells wereseeded at 5×10⁵ per 100-mm dish in medium B (Dulbecco's modified Eaglemedium containing 17 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid at pH 7.4, 3 ug/ml proline, 100 U/ml penicillin, and 100 ug/mlstreptomycin) supplemented with 10% fetal calf serum. On the followingday, the cells were transfected by the calcium phosphate coprecipitationtechnique (van de Eb et al. (1980), Meth. Enzymol., 65:826) with one ortwo test plasmids (7.5 ug of pLDLR-CAT with or without 2.5 ugpHSVTK-CAT) together with 0.5 ug pSV3-Neo. The cells were incubated withthe DNA for 5 hr and then exposed to 20% (v/v) glycerol in medium B for4 mind. Thereafter the cells were incubated in medium B supplementedwith 10% fetal calf serum for 24 hr and then switched to the same mediumcontaining 700 ug/ml G418. Selection with G418 was maintained untilstable resistant colonies could be discerned (2-3 weeks). Resistantcolonies Were pooled (150-600 per transfection), expanded in masscultures in the presence of G418 (700 ug/ml), and used for experiments.In the experiments described in FIG. 4-6, a single-cell derived subclonewas obtained by limiting dilution from a pooled cell line derived aftertransfection with pLDLR-CAT 1563, expanded in mass culture, and screenedfor CAT enzyme activity (Gorman et al., supra). A subclone expressingthe highest level of CAT enzyme activity was then examined forregulatory activity using a primer extension assay for mRNA levels (seebelow).

Sterol-Regulation Experiments

Pooled or cloned cell lines were seeded at 2×10⁵ cells per 100-mm dishon day 0 in medium A supplemented with 10% fetal calf serum. In thestandard protocol, on day 2 the cells were washed with 5 mlphosphate-buffered saline and fed with 8 ml of medium A containing 10%calf lipoprotein-deficient serum in place of whole fetal calf serum.This medium contained either no additions (induction medium) or amixture of cholesterol and 25-hydroxycholesterol in a ratio of 20:1added in 4 to 26 ul ethanol (suppression medium). On day 3 afterincubation for 20 hr in induction or suppression medium, the cells from12 dishes were harvested in 4M guanidinium thiocyanate containing 6.25g/l lauroyl sarcosine, 9.25 g/l sodium citrate, and 0.7% (v/v)B-mercaptoethanol, and the total RNA was purified by centrifugationthrough CsCl. The RNA pellet was dissolved in buffer containing 10 mMTris-chloride and 1 mM EDTA at pH 8.0 (TE buffer), precipitated withethanol, and then quantitated by OD²⁶⁰. Approximately 20-40 ug of totalRNA were obtained from each 100-mm dish of cells.

Primer Extension Assays

To detect transcripts containing CAT sequences, (derived from pLDLR-CATand/or pHSVTK-CAT), we used an mRNA-complementary primer of 40 ntcorresponding to bases 400 to 439 of the published CAT gene sequence(Alton et al. (1979), Nature, 282:864). Transcripts from the neo gene(conferring G418 resistance) were detected with an mRNA-complementaryprimer of 37 nt corresponding to bases 1407 to 1443 of the transposonTn5 gene sequence (Beck et al. (1982), Gene, 19:327). Endogenous hamsterTK mRNA was detected With a 43-nt long primer derived from bases 198 to240 of the cDNA sequence (Lewis (1986), Mol. Cell. Biol., 6:1998).Endogenous hamster HMG CoA synthase mRNA was measured by extension witha 40-nt long primer corresponding to bases 41 to 80 of the cDNA sequence(Gil et al. (1986), J. Biol. Chem., 261:3710). Endogenous hamster LDLreceptor mRNA was detected with an oligonucleotide primer of 36 nt whosesequence was derived from exon 4 of the hamster gene.

Each oligonucleotide was 5' end-labeled to a specific activity of >5000Ci/mmol with [gamma-³² P]ATP and T4 polynucleotide kinase. Primers forthe neo gene and the endogenous TK gene were diluted with an appropriateamount of unlabeled oligonucleotide to obtain a signal thatapproximately equal in intensity to that from the test plasmid. Thelabeled primers (1-2 ul of a 5-10×10⁻⁴ OD₂₆₀ units/ml solution) werecoprecipitated with 20 ug of total RNA in ethanol and resuspended in 10ul of TE buffer and 0.27M KCl for hybridization. Hybridization wascarried out for 15 min at 68˜C, after which the samples werecentrifugated for 5 sec at 4˜C. A solution (24 ul) containing 17 Ureverse transcriptase (Life Sciences, St. Petersburg, Fla.), 20 mMTris-chloride (pH 8.7), 10 mM MgCl₂, 10 mM dithiothreitol, 0.4 mM dNTPs,and 0.25 ug/ml actinomycin D was added to each tube. The samples wereincubated 1 hr at 42˜C, diluted to 200 ul with TE buffer, extracted with200 ul of phenol-chloroform, and ethanolprecipitated. Samples wereresuspended in 8 ul of TE buffer, and 12 ul of a formamide-dye solutionwas added. Follow heating for 8 min at 90-100˜C and chilling on ice, thesamples were electrophoresed for 2-3 hr at 300 V on 5% polyacrylamide/8Murea gels. The gels were fixed in 10% and then 1% trichloroacetic acidfor 9 min each before drying in a heated vacuum dryer. ³² P-LabeledHaeIII-digested OX174 DNA or MspI-digested pBR322 DNA was used asmolecular weight standards. The dried gels were used to expose KodakXAR-5 film with intensifying screens at -70˜C. Densitometry wasperformed on a Model GS 300 Scanning Densitometer from Hoefer ScientificInstruments.

Exemplary Experiments

In describing the LDL receptor promoter, nucleotide (nt) +1 is assignedto the A of the translation initiation codon (ATG). This convention isemployed because multiple transcription start sites located betweenpositions -93 and -79 have been identified, and thus +1 can not be usedto refer to a single site of transcription initiation (FIG. 1). The LDLreceptor sequences in each of the three plasmids, as shown in FIG. 2,terminated at the same 3' position (-58) which is located within thetranscribed region of the gene. The LDL receptor fragments were insertedinto the unique HindIII site of pSVO-CAT, a recombinant plasmid thatcontains the beta-lactamase gene, the origin of replication from pBR322,and the coding sequence for CAT (see Gorman et al. (1982), Mol. Cell.Biol., 2:1044). All of the cloning junctions in the resulting series ofLDL receptor-CAT genes (FIG. 2) were verified by DNA sequence analysisand restriction endonuclease mapping.

The 5' ends of the inserted LDL receptor gene fragments extended todifferent positions upstream (-6500, -1563, and -234). The plasmids wereintroduced into CHO cells by calcium phosphate-mediated transfectiontogether with a second plasmid containing the gene for neomycin (G418)resistance linked to the SV40 early region promoter. PermanentG418-resistant colonies were selected, and pools of approximately 150 to600 colonies from each transfection experiment were assayed forexpression of LDL receptor-CAT mRNA by primer extension. The cells wereincubated for 24 hr either in the absence of sterols (induction medium)or in the presence of a mixture of cholesterol and 25-hydroxycholesterol(suppression medium). This sterol mixture was used because it is morepotent than cholesterol alone in suppressing the LDL receptor as well asother sterol-repressed genes.

Cells transfected with each of the three LDL receptor-CAT plasmidsproduced a fusion mRNA that initiated in the receptor cap region asdetermined by primer extension with a ³² P-labeled oligonucleotidespecific for the CAT coding sequence (FIG. 3A). To assay forsterol-specific suppression of the LDLR-CAT mRNA, the amount of mRNAproduced by the co-transfected neo gene was determined simultaneously.The expression of this gene is driven by the constitutive SV40 earlyregion promoter and does not respond to sterols. When the transfectedCHO cells were grown in the presence of sterols, the amount of mRNAtranscribed from the various LDL receptor-CAT genes was suppressed by50-83% relative to the amount of neo mRNA (FIG. 3A). To ensurereproducibility of these assays, they were repeated on three differentlines of CHO cells that were transfected with the pLDLR-CAT constructson three separate occasions; all cell lines gave similar results. FIG. 3shows the results with two separate -234 constructs. These data showthat the most important sequences for expression and sterol regulationof the LDL receptor gene are contained within the 177-bp fragmentextending from position -234 to -58. Identical results were obtainedwhen these same LDLR-CAT genes were transfected into an SV40-transformedline of human fibroblasts, human epidermoid carcinoma A431, mouse Y1adrenal cells, and mouse L cells (data not shown). These resultsindicate the general applicability of the LDL receptor promoter andregulatory sequences.

Using the same mRNA samples as those in FIG. 3A, the amount of LDLreceptor mRNA derived from the endogenous hamster receptor gene wasestimated (FIG. 3B). For this purpose, an oligonucleotide primer wasused that is complementary to mRNA sequences encoded by exon 4 of thehamster gene that are located about 575 nt 3' to the cap site. The useof such a remote oligonucleotide primer was necessary because to dateonly sequences corresponding to exons 4 through 18 of the hamster LDLreceptor gene have been isolated by the present inventors.

In extending over the large distance separating the primer and the 5'end of the mRNA, the reverse transcriptase encountered several strongstop sites. As a result, a family of primer-extended products wasgenerated (far right lane, FIG. 3B). The most abundant extensions aremarked by the black dots in FIG. 3B. In the presence of sterols, all ofthese primer-extended products were reduced in amount. On the otherhand, the primer-extended product corresponding to mRNA derived from thetransfected neo gene was not suppressed (FIG. 3B). The relative amountof LDL receptor mRNA suppression was estimated by densitometric scanningof the band corresponding to the full-length prime-extended product(band c) and by comparing it to the primer-extended product of the neogene (band a). The results showed that sterols suppressed the endogenousLDL receptor mRNA by 52-81% in the various cell lines (FIG. 3B), adegree of suppression that was similar to that observed for thetransfected human LDL receptor promoter-CAT gene (50-83%, FIG. 3A).

FIG. 4 shows an experiment designed to compare the sensitivity of thetransfected LDLR-CAT promoter and the endogenous LD receptor promoter toincreasing concentrations of sterols. For this purpose, the constructthat extended to position -1563 in the LDL receptor 5' flanking region(pLDLR-CAT-1563 of FIG. 2) was used. As a control, another ³² P-labeledoligonucleotide primer was employed to measure the amount of cellularmRNA for thymidine kinase (TK) produced by the endogenous hamster TKgene. The addition of 10 ug/ml cholesterol plus 0.5 ug/ml of25-hydroxycholesterol produced a nearly complete suppression of theLDLR-CAT mRNA without affecting the level of endogenous TK mRNA (FIG.4A). This concentration of sterols strongly suppressed the endogenousLDL receptor mRNA in the same cells black dots (FIG. 4B). The amount ofmRNA for another hamster cholesterol-suppressed gene, HMG CoA synthase,was also measured using a ³² P-labeled oligonucleotide primercomplementary to the synthase mRNA. This primer produced two extendedproducts which reflect the existence of two species of synthase mRNAthat differ in the presence or absence of a 59-bp optional exon in the5' untranslated region. Both of these transcripts were suppressed bycholesterol plus 25-hydroxycholesterol (FIG. 4B).

FIG. 5 summarizes in graphic form the quantitative results of theexperiment shown in FIG. 4. These data show that the endogenous LDLreceptor mRNA and the mRNA derived from the LDLR-CAT 1563 construct weresuppressed in parallel by the sterol mixture. On the other hand,endogenous HMG CoA synthase mRNA was more sensitive to the sterols.Complete suppression of this mRNA occurred at 3 ug/ml cholesterol plus0.3 ug/ml 25-hydroxycholesterol.

To determine the time course of induction of the mRNA derived frompLDLR-CAT 1563 following the removal of sterols from the medium, theexperiment shown in FIG. 6 was performed. Cells were maintained insuppression medium containing 10 ug/ml cholesterol and 0.5 ug/ml25-hydroxycholesterol for 20 hr and then switched to induction medium(no sterols) for varying time periods. Total cellular RNA was thenisolated and subjected to primer extension analysis usingoligonucleotides specific for the LDLR-CAT mRNA and the endogenoushamster TK mRNA. The amount of LDLR-CAT mRNA rose 4-fold by 2 hr aftersterol removal and reached a maximum (8-fold) at 11 hr (FIG. 6). Asexpected, there was no change in the level of the endogenous TK RNA.

The experiments of FIGS. 3-6 indicate that fragments of the LDL receptorpromoter that include sequences from -234 to -58 are capable of drivingexpression of the CAT gene in a sterol-responsive manner. To delineatefurther sequences within this 177-bp fragment that confer positive andnegative regulation, the series of 15 promoter mutations shown in FIGS.7 and 8 were constructed and analyzed. To avoid problems associated withgross deletions, individual overlapping 10-bp segments of the promoterfragment from pLDLR-CAT 234 were scrambled by site-directed mutagenesis(FIG. 7). Each mutant promoter was then transfected into CHO cells ontwo to five separate occasions, together with pSV3-Neo for G418selection and pHSVTK-CAT as a transfection control. The latter plasmidcontains TK promoter sequences (from -108 to +55) derived from the HSVgenome fused to the CAT gene. The HSV TK promoter has been wellcharacterized by McKnight and coworkers (McKnight et al. (1982) Cell,31:355; McKnight et al. (1982), Science, 234:47) and does not respond tosterols. By comparing the amount of mRNA derived from the HSVTK-CAT geneto that from a transfected LDLR-CAT gene, the relative promoterstrengths of the different LDL receptor mutations was estimated and the"% suppression" obtained in the presence of sterols calculated. Theresults from one primer extension analysis are shown in FIG. 8.

RNA was isolated from CHO cells transfected with pLDLR-CAT 234 andpHSVTK-CAT and grown in the absence of sterols. When this RNA wassubjected to primer extension using CAT-specific and endogenous hamsterTK-specific ³² P-oligonucleotides, three products were visualized afterautoradiography (FIG. 8, left lane of upper panel). Two products werederived from the transfected chimeric CAT genes; the 314-nt band is frommRNA initiated at the correct cap site of the HSVTK-CAT gene, while the290-nt band is the product of the LDLR-CAT gene. The third product is a260-nt band that corresponds to the primer extension product from theendogenous TK gene. The addition of sterols to the medium resulted in an83% reduction in the amount of the LDLR-CAT mRNA relative to theHSVTK-CAT mRNA (FIG. 8, second lane of upper panel). A similar reductionwas calculated using the endogenous TK mRNA as a standard (data notshown).

Experiments with the mutated LDL receptor-CAT genes yieldedqualitatively similar results with respect to sterol regulation. Nine ofthe mutated promoters produced an mRNA whose transcription wassuppressed in a normal manner in response to sterols. One mutation(-186/-177) responded less well to sterols, suppressing transcription ofthe LDLR-CAT mRNA by only 25% (FIG. 8). However, in this case theoverall transcription of the mutant gene was substantially reduced,making it difficult to accurately measure suppression.

Many of the scramble mutations dramatically affected positive expression(FIG. 8). To compare relative transcription levels between the mutantgenes, a value of 1.0 was assigned to the amount of mRNA transcribedfrom the transfected normal LDLR-CAT 234 gene in the absence of sterols(b) divided by that from the HSVTK-CAT gene (a). A similar ratio wascalculated from each of the scramble mutations based on the data shownin FIG. 8 and on two to four additional experiments. These ratios areplotted in histogram form in FIG. 9, where the ordinate representsrelative transcription and the abscissa represents DNA sequences fromthe human LDL receptor promoter. This data indicate that mutations thatscramble sequences residing in any of the three direct repeats reducetranscription 50 to >95%. Similarly, a mutation that disrupts the more5' of the two TATA sequences decreases transcription by more than 90%(FIG. 9). Several of the mutations increased transcription moderately(approximately 2-fold). Surprisingly the mutation that scrambled themore 3' TATA sequence (-109/-100) increased the amount of LDLR-CAT mRNAsome 29-fold. This number probably represents an overestimate of therelative promoter activity of the -109/-100 mutation in that thecotransfected HSVTK-CAT gene to which it was compared was transcribedpoorly in these cells (see FIG. 8). Identical results were obtainedusing RNA from two separate transfections with this mutant indicatingthat the reduced HSVTK-CAT transcription was not due to differentialtransfection of the two plasmids.

EXAMPLE I OVERVIEW

The data presented by the foregoing experiments define a minimal DNAregion from the human LDL receptor gene to which expression andsterol-dependent regulation functions may be ascribed, at least in thecontext of the LDL receptor gene per se. When fused to a bacterialmarker gene and transfected into CHO cells, 5'-flanking DNA from thereceptor gene directed the synthesis of a correctly initiated mRNA thatwas decreased in amount when sterols were added to the medium (FIG. 3).Titration experiments revealed that the response to exogenously addedsterols was equivalent for both a transfected gene having human receptorsequences between -1563 and -58 and the endogenous hamster LDL receptorgene (FIG. 5). The kinetics of induction of this mRNA were rapid;half-maximal expression was obtained 2 hr after removal of sterols (FIG.6). These results indicate that the turning on of the LDL receptorpromoter signals when sterols are removed from the media is very rapid.

By further reducing the amount of human receptor DNA in the fusion gene,a segment spanning sequences between -234 and -58 was found to besufficient for the expression of a sterol-responsive mRNA. The amount ofmRNA transcribed from this construct and its sterol response wereidentical to mRNAs synthesized from the chimeric genes containing largeramounts of 5'-flanking receptor DNA, indicating that no importanttranscription signals for regulation and expression had been deleted(FIG. 3).

The 177-bp fragment of receptor DNA in pLDLR-CAT 234 was small enough toallow a further delineation of transcriptionally important sequences bya form of saturation mutagenesis. To this end, 15 mutations wereintroduced by site-directed oligonucleotide mutagenesis in whichoverlapping 10-bp segments were scrambled. The results from transfectionexperiments with these plasmids indicated that all three of the 16-bpdirect repeats in the receptor promoter are required for maximalexpression (FIG. 9). In addition, the more 5' of the two TATA-likesequences (TTGAAAT) was required for maximal expression. Surprisingly, amutation which scrambled sequences in the 3' TATA-like sequence(TGTAAAT) led to a marked increase in transcription from the mutant gene(FIG. 9). The mechanism behind this promoter-up phenotype is at presentnot known, although it was noted that the mutation did not alter thestart site of transcription, as an identical primer extension product isobtained with mRNA from cells transfected with this construct (FIG. 8).Neither of these TATA-like sequences match well the canonical sequenceTATAAA derived from many other eukayrotic genes. Thus, it is conceivablethat the more 3' element may play more of a regulatory role (as observedhere) than as a signal for precise mRNA start site selection as observedin other genes. Future studies with the pLDLR-CAT -109/-100 constructshould clarify the role of this sequence.

With respect to the direct repeats, mutations that alter repeat 1decrease transcription to a slightly lesser extent (50-90%) than thosethat alter repeats 2 and 3 (80-95% decrease). These differences may bereal, implying non-equivalence of the three repeats with respect toexpression, or they may be a consequence of the exact sequence scrambledin a given repeat. In this light, it is notable that a mutation thatalters as few as 3 bp of a direct repeat leads to decreasedtranscription: mutation -203/-194 alters the 5'-three nucleotides ofrepeat 1 and reproducibly decreased mRNA synthesis 50% (FIG. 8). Theseresults imply that each direct repeat sequence is recognized essentiallyin its entirety by a transcription factor or factors.

In considering which of the known transcription factors might interactwith these sequences, it was discovered that the central core of the LDLreceptor direct repeats shares sequence homology with the so-called "GCboxes" found in other eukayrotic RNA polymerase II promoters (Kadonagaet al. (1986), Trends Biochem. Sci., 11:20). Table 1, below, indicatesthat repeats 1, 2 and 3 have 8 of 10, 7 of 10, and 9 of 10 matches,respectively, with a consensus GC box sequence. Tijan and colleagueshave recently isolated in homogeneous form a protein termedtranscription factor Spl (Briggs, e al. (1986), Science, 234:47) andhave shown that in several viral and cellular promoters this proteinstimulates transcription by binding to GC sequences (Dyan et al. (1985),Nature, 316:774). Initially, it was postulated that Spl had arecognition sequence of 10 nucleotides with a central core consisting ofCCGCCC, which is not found in any of the three direct repeats of the LDLreceptor promoter (Table II).

                  TABLE II    ______________________________________    GC Box Homologies in the Direct Repeats    of the Human LDL Receptor Promoter    ______________________________________    Repeat 1                  ##STR2##    Repeat 2                  ##STR3##    Repeat 3                  ##STR4##    GC Box Consensus*                  ##STR5##    ______________________________________     Nucleotides in repeats 1, 2 and 3 that differ from the GC box consensus     sequence are underlined.     *From Kadonaga et al. (1986), Trends Biochem. Sci., 11:20-23.

More recently, two decanucleotide sequences that differ from the Splconsensus sequence by 2 positions in the canonical CCGCCC, have beenshown to bind Spl and activate transcription from the humanimmunodeficiency virus (HIV) long terminal repeat promoter (Jones et al.(1986), Science, 230:511). This observation suggests to the presentinventors that there is some flexibility for deviation from the Splconsensus sequences and raises the possibility that the sequences inrepeats 1 and 3 of the LDL receptor, which differ by 1 and 2 positions,respectively, from CCGCCC, are in fact Spl binding sites (Table II). Inthis regard, repeats 1 and 3 of the LDL receptor promoter bind a proteinpresent in HeLa cell nuclei which protects these sequences fromdigestion with DNAase I (see Example II). The protected regions spanabout 20 bp each, which is similar in size to protection obtained withhomogeneous Sp1.

The studies disclosed below in Example II demonstrate that directrepeats 2 and 3 of the human receptor gene function as a translocatablesterol regulatory element (SRE). For example, in these studies it isshown that the insertion of a 42-bp fragment spanning these two repeatsinto the promoter of the HSVTK gene confers negative regulation bysterols on the expression of the chimeric gene. The studies in thepresent example show that in addition to harboring an SRE sequence,these two direct repeats also contain positive transcription signals.Furthermore, none of the scramble mutations analyzed in FIG. 8 led to aconstitutive promoter, indicating that the sterol-regulatory andpositive expression sequences of the LDL receptor promoter areintimately related. If these signals are the same, it is conceivablethat competition for binding between a sterol repressor and Spl, or anSpl-like transcription factor, to a direct repeat sequence may underlythe ability of sterols to repress transcription from the LDL receptorgene. Future studies centered around the purification of the proteinsthat interact with repeats 2 and 3 may provide support for thishypothesis.

EXAMPLE II Isolation and Characterization of the LDL Receptor SterolRegulatory Element

In Example I, the expression of chimeric genes containing varioussequences from the 5' flanking region of the LDL receptor gene (rangingat the 5' end from -6500 to -234 and terminating at position -58) fusedto the CAT gene were analyzed. The results showed that a 177-bp sequencefrom the receptor promoter (-234 to -58) is capable of drivingexpression of the CAT gene in a sterol-responsive manner. In the presentexample, experiments are presented which demonstrate that all threedirect repeats are not required in order to confer sterol-responsivetranscription to heterologous genes. In general, the followingexperiments surprisingly demonstrate that a functionally translocatableSRE resides within a 42 base pair sequence which contains repeats 2 and3, but not repeat 1, and which are solely responsible for conferringsterol responsivity to heterologous structural genes. Furtherexperimentation conducted by the present inventors has demonstrated thatthe SRE is contained solely within repeat 2.

SELECTED MATERIALS AND METHODS EMPLOYED FOR EXAMPLE II

Materials were obtained from those sources listed above in Example I.Plasmids were constructed by standard techniques of genetic engineering(Maniatis, et al., (1982)) and verified by DNA sequence analysis andrestriction mapping. Plasmid A was derived from pTKdelta32/48 ofMcKnight and Kingsbury (1982), Science, 217:316, and pTK-CAT of Cato, etal. (1986), EMBO J., 5:2237. Plasmids B through D were constructed byligating the indicated LDL receptor promoter sequences (synthesized onan Applied Biosystems Model 380A DNA Synthesizer) into the HindIII-BamHIsites at -32 of the truncated HSV TK promoter in plasmid A. Plasmid E,which contains HSV TK sequences from -60 to +55, was constructed frompTK-CAT (Cato, et al., supra) and pTKdelta60/80 (McKnight and Kingsbury,supra). Plasmids F through H were engineered by ligating synthetic LDLreceptor sequences into the HindIII-BamHI sites at -60 of the truncatedHSV TK promoter.

Plasmids I-N contained the entire active HSV TK promoter extending from-480 through +55 except for the sequence between -48 and -32, which wasreplaced with short sequences corresponding to various parts of thesterol regulatory element of the LDL receptor promoter. The startingplasmid (I) was engineered from pTK-CAT (Cato, et al, supra) and plasmidLS-48/-32 of McKnight 1982), Cell., 31:355. Plasmid I thus contains anactive HSV TK promoter (with a 10-bp BamHI linker replacing viralsequences between -48 and -32) linked to the CAT gene. To constructplasmids J, K, and M, a pair of complementary oligonucleotides 42 basesin length (see Table III) were synthesized on a Model 380A DNAsynthesizer, annealed, phosphorylated at their 5' ends using ATP and T4polynucleotide kinase, and ligated into BamHI-cleaved plasmid I. Thethree desired plasmids containing varying numbers and orientations ofthe 42-mers were then identified by restriction mapping and DNAsequencing. Plasmids I and N and 0 through R were constructed in asimilar manner except that oligonucleotides of different sequences(Table III) were employed in the ligation.

DNA Transfection. CHO-KI cells were cultured, transfected with plasmids,and selected with G418 as described by Davis, et al. (1986), J. Biol.Chem., 261:2828. After 2-3 weeks of selection, resistant colonies werepooled (300-600 per transfection), expanded in mass cultures in thepresence of G418 (700 ug/ml), and used for experiments.

Sterol-Regulation Experiments. Pooled cell lines were seeded at 2×10⁵cells per 100-mm dish on day 0 in medium A supplemented with 10% fetalcalf serum. In the standard protocol, on day 2 the cells were washedwith 7 ml phosphate-buffered saline and fed with 8 ml of medium Acontaining 10% calf lipoprotein-deficient serum in place of whole fetalcalf serum. This medium contained either no additions (induction medium)or a mixture of cholesterol and 25-hydroxycholesterol in a ratio of 20:1added in 4-26 ul ethanol (suppression medium). On day 3 after incubationfor 20 hr in induction or suppression media, the cells from 12 disheswere harvested in 4 M guanidinium thiocyanate containing 6.25 g/1lauroyl sarcosine, 9.2g/1 sodium citrate, and 0.7% (v/v)2-mercaptoethanol, and the total RNA was purified by centrifugationfugation through CsCI (Chirgwin, et al., (1979), Biochemistry, 18:5294).The RNA pellet was dissolved in buffer containing 10 mM Tris-chlorideand 1 mM EDTA at pH 8.0 (TE buffer), precipitated with ethanol, and thenquantitated by OD₂₆₀.

Primer Extension Assays. To detect transcripts containing CAT sequences,we used an mRNA-complementary primer of 40 nt corresponding to bases 400to 439 of the published gene sequence (Alton and Vapnek (I1979), Nature,282:864). Endogenous hamster TK mRNA was detected with a 43-nt longprimer derived from bases 198 to 240 of the published cDNA sequ®nce(Lewis, (1986), Mol. Cell. Biol., 6:1978). Endogenous hamster HMG CoAsynthase mRNA was measured by extension with a 40-nt long primercorresponding to bases 41 to 80 of the cDNA sequence (Gil, et al.,supra). Endogenous hamster LDL receptor mRNA was detected with anoligonucleotide primer of 36 nt whose sequence was derived from exon 4of the hamster gene (unpublished observations). ³² P-End labeledoligonucleotide primers were hybridized with 20 ug total RNA andextended according to a protocol modified from McKnight and Kingsbury(1982) (see Example I). The extension products were analyzed on 5%acrylamide/8M urea gels. After electrophoresis gels were fixed and driedbefore being exposed to intensifying screens at -70° C. Densitometry wasperformed on a Hoefer scanning densitometer (Model GS-300).

Exemplary Studies

FIG. 1 shows the nucleotide sequence of the coding strand of the LDLreceptor gene in this general region. The cluster of transcription startsites at positions -93 to -79 is indicated. The prominent features ofthis region include two AT rich sequences (-116 to -101) that maycontain the equivalent of a TATA box. To the 5' side of this region,there is a segment that contains 3 imperfect direct repeats of 16 bp,two of which are in immediate juxtaposition. These repeats are alignedat the bottom of the figure.

Sterol-Mediated Suppression of LDL Receptor HSV TK Genes

Three overlapping fragments of the LDL receptor promoter weresynthesized and linked to HSV TK sequences extending from -32 to +55(plasmids A-D, FIG. 10 or from -60 to +55 (plasmids E-H, FIG. 10). HSVTK sequences between -32 and +55 contain the TATA box and cap site ofthis viral gene, whereas sequences between -60 and +55 also include thefirst upstream promoter element (GC box). These LDL receptor-HSV TKplasmids were introduced into CHO cells by calcium phosphate-mediatedtransfection together with a second plasmid containing the gene forneomycin (G418) resistance. Permanent G418-resistant colonies wereselected, and pools of 300-600 colonies were assayed for expression ofLDL receptor-CAT mRNA by primer extension. The cells were incubated for24 hr either in the absence of sterods (induction medium) or insuppression medium that contained a mixture of cholesterol and25-hydroxy-cholesterol. cholesterol. As a control for specificity ofsuppression, the amount of endogenous hamster TK mRNA was measured. Toestablish a baseline of expression for comparative purposes, the ratioof the amount of mRNA produced by the transfected gene divided by thatproduced by the endogenous hamster TK gene (as determined bydensitometric scanning of the bands was calculated).

The fragment of the TK promoter extending to position -32 includes theTATA box but lacks two upstream elements necessary for transcription. Asexpected, this plasmid (plasmid A) did not produce detectable amounts ofCAT mRNA in the CHO cells. When a fragment of the LDL receptor extendingfrom position -235 to position -124 was placed in front of the TK 32promoter fragment, detectable amounts of a mRNA which initiated at theHSV TK cap site were produced (plasmid B). The amount of this mRNA wasreduced by 60% when sterols were present. A similar effect was seen whena smaller fragment of the LDL receptor (extending from -199 to -124) wasused (plasmid C). On the other hand, when a fragment encompassingsequences between -235 and -162 (which lacked repeats 2 and 3, butincluded repeat 1 of the LDL receptor promoter) was fused to the TK 32DNA (plasmid D), only trace amounts of a CAT mRNA were produced, andthere was no suppression by sterols. Comparable results were obtainedwhen the same fragments of the LDL receptor promoter were linked toposition -60 of the HSV TK gene just to the 5' side of its firstupstream promoter element (plasmids E-H in FIG. 10). The relativeamounts of suppression by sterols were similar in both cases.

The data shown in FIG. 10 indicate that fusion of a DNA fragmentcontaining all three 16-nt direct repeats of the LDL receptor promoterto either HSV TK-CAT gene (TK 32 or TK 60) results in thesterol-regulated expression of correctly initiated mRNAs (plasmids D andH). However, insertion of a fragment containing only the first directrepeat led to very little transcription of the fusion gene, andregulation was not observed. These studies demonstrate that repeats 2and 3 contain both positive and negative elements of sterol-regulatedexpression.

Fusion Genes Containing LDL Receptor SRE Linked to HSV TK

To further evaluate the role of direct repeats 2 and 3 insterol-mediated suppression, synthetic oligonucleotides corresponding tothese sequences were prepared and inserted into an HSV TK promotercontaining a BamHI linker between positions -48 and -32 (FIG. 11). Incontrast to the earlier constructs, this HSV TK promoter retains allthree signals required for expression of the viral gene. Previousstudies by McKnight ((1982), Cell., 31:355) have shown that theinsertion of 42 bp at the position of the BamHI linker in this HSV TKpromoter reduces transcription moderately. Insertion of longer fragments(>50 bp) eliminates the promoter activity of this DNA.

A synthetic DNA fragment of 42 bp (designated SRE 42 for sterolregulatory element of 42 bp) that contained direct repeats 2 andtogether with 5 bp of flanking sequence on both sides (Table II) wasprepared. This fragment was synthesized with BamHI compatible stickyends and ligated into the BamHI linker of pHSV TK-CAT. Aftertransfection and primer extension analysis, the results shown in FIG. 11were obtained. The starting construct with the BamHI linker producedmeasurable amounts of a correctly initiated mRNA (plasmid I) asexpected. There was no suppression of transcription when sterols wereadded to the medium. Insertion of the LDL receptor SRE 42 within theBamHI linker (plasmid J) also led to the synthesis of the appropriatemRNA. However, when these cells were incubated with sterols, the amountof this mRNA declined by 57%. When the SRE 42 sequence was inserted inan orientation opposite to that found in the LDL receptor (plasmid K),the expected mRNA was still transcribed. Moreover, the amount of mRNAwas reduced by 84% when sterols were added. To control for sequencespecificity of the 42-bp SRE, the sequence was scrambled into a randomorder without changing its base composition or length (Table II). Whenthis scrambled sequence was inserted at the BamHI linker of the HSV TKpromoter, it abolished transcription (plasmid L) (FIG. 11).

                                      TABLE III    __________________________________________________________________________    LDL Receptor SRE Fragments    Synthetic DNA Fragment                  Sequences.sup.a                       Plasmid.sup.b    __________________________________________________________________________     SRE 42                  ##STR6##                               J,K,M    Scramble 42                  ##STR7##                              L,N    Footprint 3                  ##STR8##                              O,P    Single repeat 3                  ##STR9##                              Q,R    __________________________________________________________________________     .sup.a Nucleotides identical to the LDL receptor promoter are capitalized     .sup.b See FIGS. 4 and 7 for plasmid descriptions.

HSV TK promoters containing two copies of the SRE 42 sequence for atotal insertion of 84 bp (plasmid M) also express a correctly initiatedmRNA and the degree of suppression by sterols (>95%) was more than thatachieved with a single copy. When an 84-bp scrambled sequence wasinserted into the HSV TK promoter (plasmid N), transcription wasabolished (FIG. 11). In all experiments using plasmids I-N, the addedsterols did not suppress endogenous CHO TK mRNA (FIG. 11). The amount ofmRNA for another hamster cholesterol-suppressed gene, HMG CoA synthasewas also measured, using a ³² P-labeled oligonucleotide primercomplementary to the synthase mRNA (FIG. 11). This primer produced twoextended products which reflect the existence of two species of synthasemRNA that differ in the presence or absence of a 59-bp optional exon inthe 5' untranslated region. Both of these transcripts were completelysuppressed by cholesterol plus 25-hydroxycholesterol; only the larger ofthe two products is shown in FIG. 11.

The HSV TK-CAT plasmid containing two copies of the 42-bp SRE from theLDL receptor gene (plasmid M) showed the same sensitivity to sterolsuppression as did the endogenous LDL receptor gene. The hybrid plasmidwas suppressed 80% at a concentration of 10 ug/ml of cholesterol plus0.5 ug/ml of 25-hydroxycholesterol, which was similar to the level atwhich the endogenous receptor promoter was suppressed in the same cells.Endogenous synthase mRNA in these cells was measured and it wassuppressed at lower levels of sterols than was the receptor mRNA.

DNAase I Footprinting of LDL Receptor Promoter - In an effort to furtherdefine the SRE of the LDL receptor, experiments were conducted toidentify by DNAase footprinting. nuclear protein factors that mightinteract with this element. Accordingly, a fragment of the LDL receptorpromoter extending from -1563 to -58 was 5' end-labeled on the codingstrand by a method described below. A ³² P-end labeled, double strandedDNA probe was synthesized by hybriding a ³² P-end labeledoligonucleotide to a complementary M13 clone containing the LDL receptorpromoter sequence (-1563 to -58). Double stranded DNA was obtained afterextension of the primer in the presence of unlabeled dNTPs and used forfootprinting after two phenol-chloroform and chloroform extractions.Footprinting was performed as described by Briggs, et al. (1986),Science, 234: 47, using HeLa cell nuclear extracts prepared by themethod of Dignam et al. (1983), Nucl. Acids Res., 11: 1475.

Four distinct protected regions, or footprints, were seen. Footprint 4was located at -116 to -101, which is in the region of the TATA-likesequences. Footprint 3 extended from -151 to -129, encompassing repeat 3plus six bp on the 3' end of repeat 2. Footprint 2 corresponded almostexactly to repeat 1 at -196 and -181. Footprint 1 corresponded to alonger sequence that was located further upstream (-250 to -219).Footprint 3 was of particular interest because it mapped within theSRE-42 sequence that had been identified as important for both promotionand suppression of LDL receptor activity in the HSV TK constructs (FIG.11). This result suggested that the 23 bp protected from DNAase Idigestion in footprint 3 might constitute the minimum amount of DNArequired for an SRE. To test this hypothesis, we prepared syntheticoligonucleotides corresponding to footprint 3 and inserted them into theTK promoter.

Fusion Genes Containing Footprint 3 or Repeat 3 of LDL Receptor PromoterLinked to HSV TK - A synthetic DNA fragment corresponding to the regionoccupied by footprint 3 (see Table III) was inserted into the BamHIlinker at position -48/-32 of the HSV TK promoter (plasmid 0, FIG. 12).In CHO cells transfected with this plasmid, a correctly initiated mRNAwas transcribed; however, there was no suppression by sterols. A similarlack of suppression was seen when the footprint 3 region was inserted inan inverted orientation (plasmid P).

To determine whether repeat 3 by itself would affect transcription in asterol-dependent manner, we synthesized a DNA fragment that containedtwo copies of repeat 3 separated by a 6-bp linker sequence (Table III).HSV TK-CAT plasmids containing these two copies in either orientation(plasmids Q and R) expressed an mRNA that initiated at the HSV TK capsite, but neither plasmid showed sterol-mediated suppression of thistranscript (FIG. 12).

Example II Overview

End-product repression of genes that control the biosynthesis ofessential substances is a well-understood homeostatic mechanism inbacterial and yeast. The regulation becomes much more complex when acell can control the uptake of the nutrient as well as its synthesiswithin the cell. How does a cell choose between external and internalsources? This balance is particularly delicate in mammalian cholesterolhomeostasis because the uptake mechanism controls not only the level ofcholesterol in cells but also the level of cholesterol in blood. Thecells of the body must express sufficient LDL receptors to ensureefficient removal of cholesterol from blood, yet they must not producetoo many receptors or cholesterol will accumulate to toxic levels withinthe cell.

In rapidly growing cultured cells such as fibroblasts, the two sourcesof cholesterol are balanced in favor of exogenous uptake. As long as LDLis available, cultured fibroblasts preferentially use the LDL receptorto obtain cholesterol and they suppress the biosynthetic pathway. Whencellular cholesterol levels decline, the cells increase the number ofLDL receptors. If these do not provide sufficient cholesterol, thepathway for cholesterol synthesis is derepressed.

The foregoing experiments identify a 42-bp sequence within the 5'-flanking region of the LDL receptor gene that conferssterol-responsivity when inserted into the promoter region of the HSV TKgene. This sequence consists of two imperfect direct repeats of 16 bp(designated repeats 2 and 3) plus a total of 10 bp of flankingsequences. Moreover, more recent experimentation have suggested to theinventors that the entire 42 bp sequence, although preferred, is notrequired for conferring sterol regulation. Rather, the 16 bases whichcomprise repeat 2 alone is sufficient. For example, the insertion ofrepeat 2 alone into the HSV TK promoter at -60, in either orientation,was found to confer sterolresponsivity responsivity on the hybridpromoter.

The 42-bp sequence is referred to as the sterol regulatory element 42(SRE 42), and is located 35 bp upstream of the most 5' transcriptioninitiation site in the LDL receptor gene (FIG. 1). The sequence betweenthe SRE 42 and the cluster of transcription start sites at AT rich andmay contain one or more TATA-like elements. About 20 bp upstream ofrepeat 2 of the SRE 42 there is an additional copy of the 16-bp sequencethat is designated repeat 1.

The activity of the SRE 42 was analyzed by inserting a syntheticoligonucleotide into the complete HSV TK promoter at position -32 (FIG.11). This construct retained all of the viral transcription elementsrequired for TK expression. Abundant transcription of these genes wasobserved, and sterol suppression was obtained when the SRE 42 waspresent in either orientation (FIG. 11). When the same nucleotides wereinserted in a scrambled fashion, no transcription was observed (plasmidI, in FIG. 11). Two copies of the SRE42 allowed transcription, and inthis case the extent of suppression by sterols was maximal. More recentexperiments have indicated that four or more repeats, inserted in eitherthe forward or reverse direction, provides an ever more effective SRE.

When the SRE sequence was scrambled, transcription was no longerobserved. Based on previous work, an insertion of 84 bp into the -32position would be expected to abolish transcription. These data suggestthat the SRE 42 contains both positive and negative elements. Thepositive element allows transcription of the TK promoter; the negativeelement allows this transcription to be repressed in the presence ofsterols.

The present invention supports a model in which a single element of 42bp contains a sequence that binds a positive transcription factor.Sterols might repress transcription by inactivating this positivetranscription factor. This mechanism would be an inverse variation ofthat used in the lac operon of E. coli in which an inducer binds to therepressor and inactivates it. Alternatively, sterols might bind to aprotein that in turn binds to the SRE 42 and prevents the positivetranscription factor from binding. This mechanism would be analogous tothe type of repression that occurs in the promoter of bacteriophage andwould be consistent with current models of steroid hormone action inmammalian cells in which steroids bind to a receptor that in turn bindsto specific sequences in DNA.

EXAMPLE III Construction of an Expression Vector for Human GrowthHormone Using a Low Density Lipoprotein (LDL) Receptor Sterol RegulatoryElement (SRE) Promoter

Human growth hormone is a polypeptide hormone responsible for mediatingnormal human growth. The absence of this protein gives rise to theclinical syndrome of dwarfism, an abnormality that affects an estimated1 in 10,000 individuals. Until recently, the sole source of growthhormone was pituitary extracts obtained in crude form from cadavers. Theisolation and expression of the gene for growth hormone by scientists atGenentech provides an alternate source of this medically valuableprotein. The present example describes the use of a DNA segmentcontaining an LDL receptor sterol regulatory element that will allow theregulated expression of the human growth hormone gene product. As theoverproduction of proteins not normally expressed in a given cell line(such as growth hormone in Chinese hamster ovary cells described here)frequently kills the host cell, the ability to use the LDL receptor SREas a "protective" on/off switch represents a profound improvement in theproduction of growth hormone by genetic engineering methods.

The construction of an expression vector using the SRE sequence of thepresent invention involves the fusion of such segments to the growthhormone gene. A strategy for making this construction is outlined below(see FIG. 13). lt is recognized that other strategies, including thoseemploying the polymerase chain reaction, could be used to accomplishthis goal.

A DNA fragment of about 610 base pairs (bp) containing the HerpesSimplex Virus (HSV) promoter with 2 copies of the LDL receptor SRE isexcised from the plasmid M of Example II (E. coli cells bearing plasmidM, (pHSVTK-SRE42), have been deposited with the ATCC on Mar. 30, 1987,and accorded ATCC designation 67376) by digestion with the restrictionenzymes HindIII and BglII. The HSV promoter contains 3 signals requiredfor its function as a promoter, these include two GC-box elementslocated at positions -100 and -60 and a TATA sequence located atposition -30. As noted in Example II, the two copies of the 42 bpfragments which contains the LDL receptor SRE have been inserted justupstream of the HSVTK TATA sequence at position -32.

A DNA fragment containing the human growth hormone (hGH) gene is excisedfrom a plasmid obtained as described in Seeburg (1982), DNA 1:239-249,by BamHI and EcoRI digestion. This 2150 bp fragment contains all fiveexons (coding sequences) of the hGH gene and a transcription terminationregion at the 3' end of the gene near the EcoRI site.

The HSVTK-LDL receptor-SRE fragment and the hGH fragment are thenligated (see FIG. 13) to the recombinant transfer vector pTZ18R(Pharmacia Corp.) previously digested with the enzymes EcoRI andHindIII. The compatible sticky ends of the various DNA fragments arejoined by the ligase under conditions described in Maniatis et al.,supra. The resulting plasmid vector, pHSVTK-SRE42-hGH, is thenintroduced into a line of cultured CHO cells by calciumphosphate-mediated DNA transfection and the appropriate clone of cellsexpressing the growth hormone gene is selected by assaying the media forimmune-reactive protein using antibodies against hGH (NationalInstitutes of Health).

The controlled expression of the hGH gene is carried out by growing theline of CHO cells containing the transfected chimeric gene in the mannerdescribed in Example II. Briefly, the cells are maintained in Ham's F12media supplemented with 10% calf lipoprotein-deficient serum,penicillin, streptomycin, and a mixture of cholesterol (10 ug/ml) and25-hydroxycholesterol (0.5 ug/ml). The latter two sterols serve to keepthe SRE element turned off, which serves to block the expression of thehGH gene. When the cells have reached confluency in the culture, themedia is switched to the Ham's F12 containing the above additions butminus the two sterols. Removal of the sterols turns the SRE element onand allows expression of the hGH at high levels. In this manner theoptimum amount of this product is generated by the cells for subsequentpurification.

EXAMPLE IV Construction of an Expression vector for Human Tumor NeorosisFactor (TNF) Using a Low Density Lipoprotein (LDL) Receptor SterolRegulatory Element (SRE) Promoter

Human tumor necrosis factor (TNF) is a protein released by mammalianmonocyte cells in response to certain adverse stimuli. The protein hasbeen shown to cause complete regression of certain transplanted tumorsin mice and to have significant cytolytic or cytostatic activity againstmany transformed cell lines in vitro. To date, the extremely low levelsof TNF released by monocytes have precluded its use as a generalanticancer agent. The recent cloning of a TNF cDNA by scientists atGenentech Corporation (EP 168, 214A) has opened the way for theapplication of recombinant DNA techniques to the generation of largequantities of this protein. Described below is the use of an expressionvector employing a powerful on/off switch embodied in the LDL receptorSRE to produce TNF in a regulated manner in Chinese hamster ovary (CHO)cells.

The construction of this expression vector employs the steps outlinedbelow (see FIG. 14).

First, a 610 base pair (bp) fragment containing two copies of the LDLreceptor SRE inserted at position -32 of the Herpes Simplex Virusthymidine kinase promoter is isolated from plasmid M of Example II (ATCCDeposit number 67376). The plasmid is first restricted with the enzymeBglII to render the DNA linear and to free the 3' end of the desiredfragment. After BglIII digestion, the resulting 4 nucleotide (nt) stickyends are made blunt-ended ended by treatment of the DNA fragment withthe DNA polymerase I Klenow enzyme in the presence of the 4deoxynucleoside triphosphates (dNTPs) as described in Maniatis et al,supra. After blunt-ending, the plasmid is restricted with the enzymeHindIII to release the 5' end of the 610 bp fragment. This fragment isgel-purified on a low melting temperature agarose gel (Maniatis et al.,supra) and held for preparation of the TNF cDNA fragment describedbelow.

A DNA fragment encompassing the complete coding region of the human TNFcDN is excised from the plasmid pTNFtrp, obtained as described in EP168,214A, in the following manner. First, the plasmid is linearized bydigestion with the enzyme XbaI and the resulting sticky ends are filledin with the Klenow enzyme in the presence of the appropriate dNTPs. Thismanipulation frees the 5' end of the TNF cDNA as a blunt-ended XbaIsite. To release the cDNA from the linearized, EcoRI-digested, filled inplasmid a second digestion with the enzyme HindIII is performed. Theresulting approximately 850 bp fragment is then gel purified on a lowmelting temperature agarose gel.

To join the HSVTK-LDL receptor SRE fragment to the TNF fragment, the twoDNAs are mixed with an equimolar amount of the recombinant transfervector pTZ18R-NotI, previously digested with HindIII, in the presence ofadenosine triphosphate and the enzyme T4 DNA ligase. This enzyme willjoin the HindIII end of the vector to that of the HSVTK-SRE fragment,and the BglIII blunt end of this fragment to the XbaI blunt end of theTNF cDNA. Finally, it will join the HindIII end of the TNF fragment tothat of the vector (see FIG. 14 ). After ligation, the DNA istransformed into E. coli cells by the calcium chloride procedure, andthe desired clones having both the HSVTK-SRE and TNF fragments areidentified and oriented with respect to the vector by colonyhybridization and restriction mapping of mini-prep plasmid DNA.

The final step in the construction of the TNF expression vectoremploying an LDL receptor SRE promoter involves the insertion of atranscription termination and poly-adenylation signal at the 3' end ofthe chimeric gene. For this purpose, a 200 bp DNA fragment containingthese signals is excised from simian virus 40 DNA by digestion with theenzymes BamHI and BclI and the resulting sticky ends are renderedblunt-ended by treatment with the Klenow enzyme and the 4 dNTPs. Thisfragment is gel purified on a low melting agarose gel and ligated intothe above intermediate vector containing the TNF gene linked to theHSVTK-SRE promoter. For this purpose, the plasmid is linearized at theunique NotI site, filled in with the Klenow enzyme, and then subjectedto ligation with the simian virus DNA fragment. After transformation ofthe DNA into E. coli, plasmids having the viral DNA in the desiredorientation are identified by restriction digestion and DNA sequencing.

To express the TNF cDNA in a regulated manner in CHO cells, the aboveexpression vector is transfected into the cells as described in ExampleII. The desired cell line is identified by a cytotoxic assay asdescribed by Pennica et al. (1984) Nature, 312:724). Once identified,the cells are grown in Ham's F12 medium supplemented with 10%lipoprotein-deficient serum, penicillin, streptomycin, and a mixture ofcholesterol (10 ug/ml) and 25-hydroxy-cholesterol (0.5 ug/ml). When thecells are grown in the presence of the two sterols, the sterols enterthe cell and inhibit the expression of the TNF gene by virtue of the5'-located SRE sequence in the HSVTK promoter. This inhibition preventsexcess TNF from accumulating in the cells or media before they havereached their apogee of growth. Once the cells have reached nearconfluency (i.e. maximum density) and are thus at their maximumproduction capabilities, the media is changed to one lacking sterols toinduce expression of the transfected TNF gene. The absence of sterolscauses a derepression of the SRE signal in the HSVTK promoter and arapid turning on of the gene. This ability to regulate the expression ofthe TNF gene will allow the production of large quantities of mediacontaining TNF in a most efficacious manner which avoids problems ofcell toxicity caused by constant over-production of a foreign proteinand problems of product (TNF) breakdown caused by proteases in the cellsand media.

EXAMPLE V Construction of an Expression vector for Human TissuePlasminogen Activator (t-PA) Using a Low Density Lipoprotein (LDL)Receptor Sterol Regulatory Element (SRE) Promoter

Human tissue plasminogen activator (t-PA) is a protein found inmammalian plasma which regulates the dissolution of fibrin clots througha complex enzymatic system (Pennica et al. (1983) Nature, 301:214-221).Highly purified t-PA has been shown to be potentially useful as an agentin the control of pulmonary embolisms, deep vein thromboses, heartattacks, and strokes. However, the extremely low levels of human t-Papresent in plasma have precluded its use as a general therapeutic agent.The recent cloning of a cDNA encoding human t-PA by scientists atGenentech Corporation (U.K. patent application 2,119,804) has opened theway for the application of recombinant DNA techniques to the generationof large quantities of this enzyme. Described below is the use of anexpression vector employing a powerful on/off switch embodied in the LDLreceptor SRE to produce t-PA in a regulated manner in Chinese hamsterovary (CHO) cells.

The construction of his expression vector employs several geneticengineering steps which are outlined below (see FIG. 15).

First, a 610 base pair (bp) fragment containing two copies of the LDLreceptor SRE inserted at position -32 of the Herpes Simplex Virusthymidine kinase promoter is isolated from plasmid M of Example II. Theplasmid is first restricted with the enzyme BglIII to render the DNAlinear and to free the 3' end of the desired fragment. After BglIIIdigestion, the resulting 4 nucleotide (nt) sticky ends are madeblunt-ended by treatment of the DNA fragment with the DNA polymerase IKlenow enzyme in the presence of the 4 deoxynucleoside triphosphates(dNTPs) as described in Maniatis et al., supra. After blunt-ending, theplasmid is restricted with the enzyme HindIII to release the 5' end ofthe 610 bp fragment. This fragment is gel-purified on a low meltingtemperature agarose gel and held for preparation of the t-PA cDNAfragment as described below.

A DNA fragment encompassing the complete coding region of the human t-PAcDNA is excised from the plasmid pT-PAtrp12 constructed as shown in U.K.Patent Application 2,119,804. First, the plasmid is linearized bydigestion with the enzyme EcoRI and the resulting sticky ends are filledin with the Klenow enzyme in the presence of the appropriate dNTPs. Thismanipulation frees the 5' end of the t-PA cDNA as a blunt ended EcoRIsite. To release the cDNA from the linearized, EcoRI-digested, filled inplasmid a second digestion with the enzyme PstI is performed. Theresulting approximately 2,000 bp fragment is then gel purified on a lowmelting temperature agarose gel.

To join the HSVTK-LDL receptor SRE fragment to the t-PA fragment the twoDNAs are mixed with an equimolar amount of the recombinant transfervector pTZI8R-NotI, previously digested with HindIII and PstI, in thepresence of adenosine triphosphate and the enzyme T4 DNA ligase. Thisenzyme will join the HindIII end of the vector to that of the HSVTK-SREfragment, and the BglIII blunt end of this fragment to the EcoRI bluntend of the t-PA DNA. In addition it will join the PstI ends of the t-PAfragment and the vector (see FIG. 15).

The final step in the construction of the t-PA expression vectoremploying an LDL receptor SRE promoter involves the insertion of atranscription termination and poly-adenylation signal at the 3' end ofthe chimeric gene. For this purpose, a 200 bp DNA fragment containingthese signals is excised from the simian virus 40 DNA by digestion withthe enzymes BamHI and BclI and the resulting sticky ends are renderedblunt-ended by treatment with the Klenow enzyme and the 4 dNTPs. Thisfragment is gel purified on a low melting agarose gel and ligated intothe above intermediate vector containing the t-PA gene linked to theHSVTK-SRE promoter. For this purpose, the plasmid is linearized at theunique NotI site, filled in with the Klenow enzyme, and then subjectedto ligation with the simian virus DNA fragment. After transformation ofthe DNA into E. coli, plasmids having the viral DNA in the desiredorientation are identified by restriction digestion and DNA sequencing.

To express the t-PA cDNA in a regulated manner in CHO cells, the aboveexpression vector is transfected into the cells as described in ExampleII. The desired cell line is identified by immunological assay asdescribed in Pennica et al., supra. Once identified, the cells are grownin Ham's F12 medium supplemented with 10% lipoprotein-deficient serum,penicillin, streptomycin, and a mixture of cholesterol (10 ug/ml) and25-hydroxy-cholesterol (0.5 ug/ml). When the cells are grown in thepresence of the 2 sterols, they enter the cell and inhibit theexpression of the t-PA gene by virtue of the 5,-located SRE sequence inthe HSVTK promoter. This inhibition prevents excess t-PA fromaccumulating in the cell or media before the cells have reached nearmaximum numbers. Once the cells have reached near confluency (i.e.maximum density) and are thus at their maximum production capabilities,the media is changed to one lacking sterols to induce expression of thetransfected t-PA gene. The absence of sterols causes a derepression ofthe SRE signal in the HSVTK promoter and a rapid turning on of the gene.This ability to regulate the expression of the t-PA gene should allowthe production of large quantities of media containing t-PA in a mostefficacious manner which avoids problems of cell toxicity caused byconstant overproduction of a foreign protein and problems of product(t-PA) breakdown caused by proteases in the cells and media.

EXAMPLE VI Construction of Screening Cell Line

pCH110 (Hall et al., J. Mol. Appl. Gen., 2:101-109 (1983)) is digestedwith NcoI and the linearized plasmid cut within the SV40 promoter isrecovered by gel electrophoresis. A synthetic oligonucleotide (SRE 42A)having the sequence ##STR10## is prepared in vitro and ligated intolinearized pCH110, transfected into E. coli 294 cells and selected onminimal plates containing ampicillin. Plasmids are isolated fromtransformant colonies. One colony harbors pCH110M, which by restrictionanalysis and sequencing is determined to contain a tandem repeat of SRE42A in the 5'-3'-5'-3' direction in relation to the direction oftranscription from the SV 40 early promoter of pCH110M.

HepG2 human liver cells (available from the American Type CultureCollection) are incubated for 4 hours with a mixture of pSV2neo(Southern et al., J. Mol. Appl. Gen., 1:327-341 (1982)), pCH110M andDEAE-dextran using the method of McCutchan et al., J. Natl. CancerInst., 41:351-356 (1968) or Sompayrac et al., Proc. Natl. Acad. Sci.USA, 78:7575-7578 (1981). Transformants are selected in Dulbecco'smodified Eagle's medium containing 10% fetal calf serum and 1.25 mg/mlG418 (Schering-Plough) (selection medium). Individual colonies resistantto G418 are picked and grown in mass culture. They are then screened forthe units of B-galactosidase activity in cell extracts according toMiller, Experiments in Molecular Genetics, Cold springs Harbor (1972),with protein concentration assayed by the Bradford procedure with bovinegamma-globulin as the standard (Anal. Biochem., 72:248-254 (1976)). Apositive clone, HepG2M, is selected which stably expressesB-galactosidase activity. Other host cells which are useful include CHOor murine tk minus cells. In addition, the cells also are transformedwith a DHFR bearing plasmid and amplified cells identified bymethotrexate selection.

An alternative construction comprises digesting pCH110 with KpnI andHpaI and recovering the B-galactosidase gene. Plasmids K or M aredigested with appropriate restriction enzymes in order to obtainlinearized plasmids from which the TK gene is deleted. TheB-galactosidase gene is ligated into these plasmids using selectedadaptors or linkers if necessary.

EXAMPLE VII Candidate Screening Assay

HepG2M is seeded into microtiter wells containing selection medium andgrown to confluence. The selection medium used for growth is exchangedfor selection medium containing a 10.5 microgram/ml cholesteroladmixture (20:1 cholesterol to 25-hydroxycholesterol by weight) in thefollowing molar ratios of cholesterol to candidate: 100,000:1, 10,000:1,1,000:1, 100:1, and 10:1. The cells are incubated for 48 hours in thepresence of the selection medium containing cholesterol admixture aspositive controls, mock treated cells as negative controls, andcholesterol:candidate proportions. Each series of wells is treated induplicate. Thereafter, the cells in each well are fixed and stained insitu for B-galactosidase activity by adding X-Gal chromogen to eachwell, allowing color to develop and screening the wells with aspectrophotometric plate reader. Candidates which enhanceB-galactosidase activity over the cholesterol repressed control areselected for further evaluation.

EXAMPLE VII Further Characterization of the SRE 1. Procedures

a. Materials

[gamma -³² P] (>5000 Ci/mmol) was obtained from ICN. Polynucleotidekinase was obtained from Pharmacia LKB Biotechnology Inc. Enzymes usedin plasmid constructions were obtained from New England Biolabs,Boehringer Mannheim, and Bethesda Research Laboratories. Reversetranscriptase was purchased from Life Sciences (AMV 007). G418 sulfate(Geneticin) was purchased from GIBCO. Newborn calf lipoprotein-deficientserum (d>1.215 g/ml) was prepared by ultracentrifugation (Goldstein etal. 1983), Meth. Enzymol., 98:241-260). Plasmid pSV3-Neo, which containsa bacterial gene that confers resistance to G418 (11), was obtained fromBethesda Research Laboratories. Plasmid pSVO-CAT (12) was kindlyprovided by Bruce Howard (National Institutes of Health). PlasmidpTK-CAT-1 contains the HSV thymidine kinase promoter (McKnight et al.(1982), Science, 217:316-324) extending 109-base pairs upstream of themRNA cap site fused to the chloramphericol acetyltransferase (CAT) gene(Sudhof et al. (1987), Cell, 48:1061-1069). Oligonucleotides weresynthesized on an Applied Biosystems model 380A DNA Synthesizer.

b. Plasmid Constructions

Standard protocols were used for all recombinant DNA technology(Maniatis et al. (1982), Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, NY., pp. 1-545). Plasmid p1471,previously referred to as pLDLR-CAT 1563 (Maniatis et al. Id. contains a1507-base pair EcoRl-Alul restriction fragment encompassing thefull-length human LDL receptor promoter (Sudlof et al. (1987), Cey,48:1061-1069) inserted into pSVO-CAT. The promoter extends 1471 basepairs upstream from the transcription start site. Plasmid pSVO-CAT is arecombinant plasmid that contains the beta-lactamase gene (amp^(R)), thepBR322 origin of replication, and the coding sequences for CAT (Gormanet al. (1982), Mol. Cell. Biol., 2:1044-1051). Plasmid pSVO-CAT has nodefined eukaryotic promoter or enhancer sequences.

To construct point mutants A-R, oligonucleotide-directed mutagenesis(Zoller et al. (1984), DNA, 3:479-488) was performed witholigonucleotides of 20 nucleotides in length containing a mismatch 10nucleotides from the 5' end. The inversion mutant pZ was made byoligonucleotide-directed mutagenesis using a 138-mer that hybridized to30 nucleotides of the wild type sequence on either side of a78-nucleotide target sequence. The 138-mer was constructed by ligatingtwo contiguous 69 mers that were each annealed to a complimentarybridging 20-mer. The 138-mer was then purified on a denaturingpolyacrylamide gel. All mutant promoters were reinserted into theHindIII site of pSVO-CAT.

c. Transfection

Chinese hamster ovary (CHO-K1 ) cells were grown in monolayer cultureand transfected wither with 0.5 ug of pSV3 Neo, 22 ug of the testplasmid, and 3 ug of pTK-CAT-1 (Protocol 1) or with 0.5 ug pSV3-Neo, 20ug of the test plasmid, and 5 ug pTK-CAT-1 (Protocol 2) by the calciumphosphate coprecipitation technique as described previously (Sudhof etal. (1987), Cell, 48:1061-1069; Sudhof et al. (1987), J. Biol. Chem.,262:10773-10779). After two to three weeks of selection for resistanceto G418 (1 mg/ml), resistant colonies were pooled (300-500colonies/transfection) and expanded in mass culture in the presence of 1mg/ml G418 and 10% fetal calf serum (Smith et al. (1988), J. Biol.Chem., 263:18480-18487). The population of stable transfectants of atest plasmid was then incubated for 24 hr in medium containing calflipoprotein-deficient serum in the absence or presence of sterols (10ug/ml cholesterol plus 0.5 ug/ml 25-hydroxycholesterol) and harvestedfor RNA analysis as previously described (Sudhof et al. (1987), Cell,48:106-1069; Sudhof et al. (1987), J. Biol. Chem., 262:10773-10779).

d. Primer Extension Assays

Primer extension assays for measurement of mRNA were carried out aspreviously described with minor modifications (Sudhof et al. (1987), J.Biol. Chem., 262:10773-10779; Gil et al. (1987), Proc. Natl. Acad. Sci.USA, 84:1863-1866). Primer was hybridized to 40 ug total cellular RNAfor 45 min at 68° C., and the extension reaction was carried out for 1hr at 42° C. Reaction products were subjected to electrophoresis on aninverted wedge gel, 40-cm in length, 1.5-mm in thickness at the negativeelectrode, and 0.4-mm in thickness at the positive electrode. To detecttranscripts containing CAT sequences (derived from pTK-CAT-1 or from atest plasmid), an oligonucleotide primer of 40 nucleotides complimentaryto bases 400-439 of the CAT mRNA (Alton et al. (1979), Nature,282:864-869) was used. Two extension products of about 315 and 316nucleotides in length are generated from pTK-CAT-1, owing to twotranscription initiation sites within the HSV thymidine kinase promoter(McKnight et al. (1982), Science, 217:316-324). These transcripts serveas an internal control for transfection efficiency and RNAquantification. A 290-nucleotide extension produce is generated fromp1471 and the various mutant derivative plasmids. Mspl restrictionfragments of ³² P-labeled pBR322 DNA were used as size standards.Quantitative densitometry was performed with a Hoefer Scanningdensitometer (Model GS300).

2. Results

Transcription assays were carried out in CHO cells that were transfectedwith plasmids containing the 5' flanking region of the LDL receptor genefused to the CAT coding sequence. All constructions used an LDL receptorfragment that extended from +36 (relative to the start site fortranscription) to -1471 (FIG. 16, upper half). The amount of mRNA in thepermanently transfected cells was measured by a primer extension assay.

Cells transfected with p1471 (containing the wild-type LDL receptorpromoter) produced relatively large amounts of mRNA when grown in theabsence of sterols (FIG. 16, right lower panel, lane 1). Forquantification, the autoradiograms were scanned densitometrically, andthe amount of mRNA produced by the LDL receptor promoter was expressedrelative to the amount produced by the HSV thymidine kinase promoter.The amount of mRNA produced by the p1471 plasmid in the absence ofsterols was arbitrarily designated as 1.0. When sterols were added, theamount of mRNA from p1471 was reduced by 4.2-fold (FIG. 16, right lowerpanel; compare lanes 1 and 2). Stated another way, there was a 4.2-foldinduction of transcription when sterols were removed from the culturemedium.

To test for the orientation dependence of the sterol regulatoryfunction, we prepared a plasmid, designated pZ, in which the segmentfrom -33 to -110 Was inverted (FIG. 16, upper half). This segmentcontains the SRE-1. As shown in FIG. 16 (lower right panel, lanes 3 and4), pZ behaved similarly to the wild-type LDL receptor promoter. Therewas a 3.8-fold induction of transcription when sterols were removed fromthe culture medium.

During the course of these experiments, some variability was noted fromone transfection to another. To be certain that the observed regulationwas not due to random fluctuations, multiple experiments were conductedwith each plasmid. The left lower panel of FIG. 16 shows the average offour experiments in which p1471 and pZ was compared. From these data, itis clear that the inversion mutant led to a slight decrease intranscription both in the absence and presence of sterols, but that thefold induction in the absence of sterols occurred independently of theorientation of the sterol-regulated element.

To test for the effect of nucleotide substitutions in the SRE-1, aseries of LDL receptor promoter plasmids were prepared in which a singlenucleotide within repeat 2 was altered (FIG. 17). All of thesesubstitutions were made within the sequence extending from -53 to -68,which includes the sterol regulatory element. Each plasmid wastransfected into CHO cells on two separate occasions, and for each apool of transfectants was studied on multiple occasions to assurestatistical reliability of the results. A sample of the raw data fromthese experiments is shown in FIG. 18, and the results are summarized inFIG. 17.

Based on the mean data from 23 experiments, the wild-type LDL receptorpromoter gave approximately a 5-fold induction in the absence ofsterols. Mutation of the promoter (-68, -67, and -66) did not alter thisregulation (FIG. 17). These plasmids showed slightly highertranscription rates than the plasmid containing the wild-type promoterboth in the absence and presence of sterols (FIG. 17A), but the foldinduction was not impaired (FIG. 17B). A major disruption of regulationwas seen when any one of the nucleotides in the TCAC sequence (-64 to-61) was altered by a transversion (change from purine to pyrimidine orvice versa). A similar decrease in regulation was seen with a transitionmutation in which the A (-62) of the TCAC was changed to G (FIG. 17B).Mutation of the C at -60 had less of an effect (FIG. 17B). The next fournucleotides CCAC (-59 to -56) were each crucial for regulation.Transversion mutations at any of these four positions essentiallyabolished the induction of transcription in the absence of sterols. Asingle transition mutation was also made in this region (A to G at -57).This mutation also abolished regulation. The final three nucleotides inrepeat 2 (TGC at -55 to -53) were not apparently important forregulation since transversion mutations had little effect (FIG. 17).

It is significant to note that none of the point mutations in FIG. 17significantly reduced transcription in the presence of sterols (FIG.17A). The mutations that abolish regulation did so by preventing theinduction in the absence of sterols.

3. General Overview of Example VIII

The 8-base pair SRE-1 was originally identified as a region of sequencesimilarity within the sterol regulatory regions of the promoters for theLDL receptor, HMG CoA synthase, and HMG CoA reductase genes (Smith etal. (1988), Jrnl. Biol. Chem., 263:18480-18487. In the LDL receptor theSRE-1 was found within the 16-base pair repeat 2 which had been shown tocontain the information necessary for sterol-mediated regulation (Sudhofet al. (1987), Cell, 48:1061-1069; Dawson et al. (1988), J. Biol. Chem.,263:3372-3379). Prior to this study, no definitive evidence wasavailable to show that the eight nucleotides comprising the SRE-1encompassed the essential element for sterol regulation. The currentstudy shows that the 8-base pair SRE-1, situated within repeat 2,carries most of the information necessary for this control. In addition,the two adjacent upstream nucleotides at positions -64 and -65 appear tobe involved.

Substitution of any single nucleotide within the 8-base pair core SRE-1markedly limited inducibility in the absence of sterols with the soleexception of the C at position -60. When this pyrimidine was changed toa purine (A), transcription remained inducible by nearly 4-fold. It isnoteworthy that in the Syrian hamster LDL receptor gene, this positionis occupied by an A. All other mutations within the SRE-1 reducedinducibility below 2.5-fold; nucleotides at these positions are allconserved in the human and hamster promoters.

In general, mutations in the tetramer at the 3' end of the core SRE-1(CCAC) had a more profound effect than mutations in the 5' tetramer(CCAC) (FIG. 17). The T at position -64 immediately adjacent to the coreSRE-1 also played a role in regulation since substitution of a G reducedthe induction. In this study the adjacent A at position -76 was notaltered. In a previous study which the repeat 2 sequence was insertedinto the HSV thymidine kinase promoter, this A was changed to a C(mutant L in Ref. 4), and this substitution reduced the amplitude ofsterol regulation by 75%. These studies thus localize the regulatoryregion of the 16-base pair repeat 2 to the central 10 base pairs. Thethree base pairs at either end of repeat 2 are not important forsterol-dependent regulation since substitutions in these positions didnot impair regulation.

None of the point mutations in the SRE-1 or other regions of repeat 2led to a constitutively high level of transcription from the LDLreceptor promoter. The only result was a failure of induction followingthe removal of sterols from the culture medium. These data stronglysuggest that in the intact LDL receptor promoter, the SRE-1 acts as abinding site for a conditionally positive activator protein thatenhances transcription in the absence of sterols and loses this effectin the presence of sterols. No evidence was found that the SRE-1 acts asthe binding site for a repressor protein. This interpretation agreeswith the conclusion drawn from studies of point mutations in both SRE-1sequences of the HMG CoA synthase promoter (Smith et al. (1988), J.Biol. Chem., 263:18480-18487). A comparative level of detail is not yetavailable for the putative SRE-1 in the HMG CoA reductase promoter.

On the basis of the current experiments with the native LDL receptorpromoter, it is concluded that the SRE-1 functions as an enhancer onlyin the absence of sterols. A working model suggests that cells contain aprotein that binds to the SRE-1 and activates transcription in theabsence of sterols and that this function is inactivated as sterolsaccumulate within the cell. Current efforts are directed toward theidentification of such a sterol-sensitive, trans-acting enhancer proteinthat demonstrates binding specificity for the SRE-1.

The current studies define the nucleotides that are required for thefunction of the SRE-1 in the context of the intact LDL receptorpromoter. Certain substitutions that are not tolerated in the LDLreceptor promoter might be tolerated in the SRE-1 sequence of othergenes. In the putative SRE-1 of the HMG CoA reductase promoter, the C atthe 5th position in the octamer (corresponding to position -59 in theLDL receptor promoter) is replaced by a G. Although this substitutiondestroys SRE-1 function in the LDL receptor promoter (FIG. 17), it mightnot have that effect in the HMG CoA reductase promoter because ofcompensatory substitutions at other nucleotides that flank the SRE-1. Inaddition, the interaction of a protein with the SRE-1 in the HMG CoAreductase promoter might be stabilized by protein-protein interactionswith neighboring DNA binding proteins so that binding of the sterolregulatory protein is not abolished by this single nucleotide change.

The foregoing examples, both actual and prophetic, demonstrateexperiments performed and contemplated by the present inventors in thedevelopment of the invention. It is believed that these examples includea disclosure of techniques which serve to both apprise the art of thepractice of the invention and, additionally, serve to demonstrate itsusefulness in a number of settings and to disseminate general knowledgewhich relates peripherally to more central aspects of the invention asdefined by the appended claims. However, it will be appreciated by thoseof skill in the art that the techniques and embodiments disclosed hereinare preferred embodiments only and that in general, numerous equivalentmethods and techniques may be employed to achieve the same result.

All of the references identified hereinabove, are hereby expresslyincorporated herein by reference to the extent that they describe, setforth, provide a basis for or enable compositions and/or methods whichmay be important to the practice of one or more embodiments of thepresent inventions.

What is claimed:
 1. A substantially purified segment of DNA comprising afunctionally translocatable sterol regulatory element capable ofconferring sterol mediated regulation of structural gene transcriptionto a selected heterologous structural gene when located upstream fromand proximal to a transcription initiation site of such a gene, providedthat said segment is free of the structural gene ordinarily under thetranscriptional control of said sterol regulatory element, the elementincluding a nucleotide sequence selected from one of thefollowing:5'-C-A-C-C-C-C-A-C-3' 5'-C-A-C-A-C-C-A-C-3'5'-C-A-C-C-G-C-A-C-3' 5'-C-A-C-C-C-T-A-C-3'or the complement of such asequence.
 2. The DNA segment of claim 1, defined further as a segment offrom 8 to 50 nucleotides in length.
 3. The DNA segment of claim 1,defined further as a segment of from 8 to 27 nucleotides in length. 4.The DNA segment of claim 1 further comprising a functionallytranslocatable positive gene promoter control element capable ofenhancing the transcription rate of such an associated structural genewhen located upstream from and proximal to such a transcriptioninitiation site.
 5. The DNA segment of claim 4 wherein said positivecontrol element comprises a nucleic acid sequence of:(a)5'-A-A-A-C-T-C-C-T-C-C-T-C-T-T-G-C-3'; (b)5'-A-A-A-C-T-C-C-T-C-C-C-C-C-T-G-C-3'; (c)5'-G-C-A-A-G-A-G-G-A-G-G-A-G-T-T-T-3'; or (d) 5'-G-C-A-G-G-GG-G-A-G-G-A-G-T-T-T-3'.
 6. The DNA segment of claim 4 wherein saidpositive control element comprises a nucleic acid sequence of:

    (X).sub.n

wherein n=1-5, each X being independently selected from DNA segmentshaving a nucleotide sequence of: (a)5'-A-A-A-C-T-C-C-T-C-C-T-C-T-T-G-C-3'; (b)5'-A-A-A-C-T-C-C-T-C-C-C-C-C-T-G-C-3'; (c)5'-G-C-A-A-G-A-G-G-A-G-G-A-G-T-T-T-3'; or (d)5'-G-C-A-G-G-G-G-G-A-G-G-A-G-T-T-T-3';with each X unit, if more thanone, being separated by from 0 to 20 nucleotides selected from the groupof nucleotides consisting of A, G, C and T.
 7. The DNA segment of claim4 comprising a nucleic acid sequence of:

    (X).sub.n

wherein n=1-5, each X being independently selected from DNA segmentshaving a nucleotide sequence of: (a)5'-A-A-A-A-T-C-A-C-C-C-C-A-C-T-G-C-A-A-A-C-T-C-C-T-C-C-C-C-C-T-G-C-3';or (b)5'-G-C-A-G-G-G-G-G-A-G-G-A-G-T-T-T-G-C-A-G-T-G-G-G-G-T-G-A-T-T-T-T-3';witheach X unit, if more than one, being separated by from 0 to 10nucleotides selected from the group of nucleotides consisting of A, G, Cand T.
 8. A DNA molecule comprising a DNA segment having a sterolregulatory element in accordance with claim 1 in combination with astructural gene not ordinarily under the transcriptional control of saidelement, said structural gene and sterol regulatory element beingcombined in a manner such that said structural gene is under thetranscriptional control of said regulatory element.
 9. The DNA moleculeof claim 8 wherein said selected structural gene comprises thestructural gene for t-PA, human growth hormone, activin, interferon,inhibin, interleukin I, interleukin II or beta-galactosidase.
 10. TheDNA segment of claim 1, including within its nucleotide sequence anucleotide sequence selected from one of the following:(a)5'-A-G-T-T-T-G-C-A-G-T-G-G-G-G-T-G-A-T-T-T-T-C-A-A-A-T-G-3'; (b)5'-A-G-T-C-T-G-C-A-G-T-G-G-T-G-T-G-A-T-T-T-T-C-A-A-A-A-G-3'; (c)5'-A-G-T-C-T-G-C-A-G-T-G-G-G-G-T-G-A-T-T-T-T-C-A-A-A-A-A-3'; (d)5'-G-A-G-A-G-A-T-G-G-T-G-C-G-G-T-G-C-C-T-G-T-T-C-T-T-G-G-3'; (e)5'-G-A-G-A-G-A-T-G-G-T-G-C-G-G-T-G-C-C-C-G-T-T-C-T-C-C-G-3'; (f)5'-A-C-G-G-T-G-A-C-G-T-A-G-G-G-T-G-G-C-C-C-G-A-G-A-A-C-A-3'; or (g)5'-C-C-G-G-G-G-A-A-G-T-G-G-G-G-T-G-A-G-A-C-G-G-T-G-A-C-G-3'; orthecomplement of any of these sequences.
 11. The DNA segment of claim 1,wherein the element comprises -C-A-C-C-C-C-A-C-, or its complement. 12.The DNA segment of claim 1, wherein the element comprises-C-A-C-A-C-C-A-C-, or its complement.
 13. The DNA segment of claim 1,wherein the element comprises -C-A-C-C-G-C-A-C-, or its complement. 14.The DNA segment of claim 1, wherein the element comprises-C-A-C-C-C-T-A-C-, or its complement.
 15. A substantially purifiedsegment of DNA comprising a functionally translocatable sterolregulatory element capable of conferring sterol mediated regulation ofstructural gene transcription to a selected structural gene when locatedupstream from and proximal to a transcription initiation site of such agene, the element including a nucleotide sequence selected from one ofthe following:a) 5'-A-A-C-C-C-C-A-C-3'; b) 5'-C-T-C-C-C-C-A-C-3'; c)5'-C-G-C-C-C-C-A-C-3'; d) 5'-C-A-A-C-C-C-A-C-3'; and e)5'-C-A-C-A-C-C-A-C-3';or the complement of such a sequence.
 16. The DNAsegment of claim 15, wherein the element comprises -A-A-C-C-C-C-A-C-, orits complement.
 17. The DNA segment of claim 15, wherein the elementcomprises -C-T-C-C-C-C-A-C-, or its complement.
 18. The DNA segment ofclaim 15, wherein the element comprises -C-G-C-C-C-C-A-C-, or itscomplement.
 19. The DNA segment of claim 15, wherein the elementcomprises -C-A-A-C-C-C-A-C-, or its complement.
 20. The DNA segment ofclaim 15, wherein the element comprises -C-A-C-A-C-C-A-C-, or itscomplement.
 21. A recombinant DNA vector comprising a DNA segment asdefined by anyone of claims 1, 2, 3, 4 through 7, 10 through 14 or 15-20or DNA molecule as defined by claim 8 or
 9. 22. A recombinant cellcomprising a recombinant DNA vector as defined by claim
 21. 23. A methodfor sterol regulating expression of a polypeptide in recombinant cellculture comprising:(a) preparing a first DNA segment as defined byanyone of claims; 4-7, or 11-14 or 15-20. (b) constructing a second DNAsegment by positioning said first DNA segment upstream from and proximalto a transcription initiation site of a selected structural gene suchthat said structural gene may be brought under the transcriptionalcontrol of the sterol regulatory element of said first DNA segment; (c)transforming a host cell with said second DNA segment; and presence of asterol in amounts and for a time sufficient to suppress transcription ofsaid structural gene.
 24. The method of claim 23 wherein said selectedheterologous structural gene comprises the structural gene for t-PA,human growth hormone, activin, interferon, inhibin, human tumor necrosisfactor, interleukin I, interleukin II or beta-galactosidase.